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Evolution of karyotypic abnormalities and C-MYC oncogene amplification in human colonic carcinoma cell lines.

Cell lines (COLO 320 DM and COLO 320 HSR), established from a human neuroendocrine tumor, contain an amplified cellular oncogene (c-myc). We have previously shown that the homogeneously staining regions (HSRs) of a marker chromosome in the COLO 320 HSR cells that evolved in culture from COLO 320 DM cells contain amplified c-myc. Molecular hybridization in situ has now been used to demonstrate that the HSRs are on both arms of what was once an X chromosome. We also show that amplified c-myc copies are present in the isolated double minute chromosomes (DMs) of the COLO 320 DM cells that were characteristic of the tumor cells initially established from the patient. The results suggest that the amplified c-myc appeared first as DMs and was subsequently transposed to engender HSRs on an X chromosome. The initial COLO 320 tumor cell may have acquired two "early replicating" (i.e., active) X chromosomes and lost the "late replicating" (i.e., inactive) X.

Cell Line↗

Induction of hemoglobin synthesis in original K 562 cell line.

Cells of the original human myelogenous leukemia cell line, K 562 were induced to synthesize hemoglobin by incubation with 0.05 mM hemin. Analysis by isoelectric focusing indicated major bands in the region of hemoglobin F, Bart's hemoglobin, and embryonics. Cytochemical analysis by the Lepehene peroxidase reaction for hemoglobin indicated an heterogeneous population of cells with respect to hemoglobin induction. Approximately 50% of the cells were positive for hemoglobin. The benzidine-positive material was present as granules in the region of the Golgi apparatus of large blasts with undifferentiated nuclei. There was no indication of normal maturation or of terminal differentiation into reticulocytes or erythrocytes.

Cell Line↗

Glycosylation and functional activity of anti-D secreted by two human lymphoblastoid cell lines.

Cell lines BTSN4 and BTSN5 were produced by the Epstein-Barr Virus (EBV) transformation of B-lymphocytes from the same human donor. Both secrete an anti-D monoclonal of the IgG1 subclass but these antibodies display vastly different effector activities. Specifically, anti-D from BTSN4 has a far greater activity in both monocyte- and lymphocyte-mediated ADCC reactions and causes a higher percentage of rosettes to be formed with monocyte-like U937 cells. This variation in functional activity is shown to coincide with changes in the structure of the sugar chains attached to the asparagine-297 site on the immunoglobulin heavy chain.

Antibodies, Monoclonal↗

Characterization of lymphoma-derived cell lines: comparison of cell lines positive and negative for Epstein-Barr virus nuclear antigen. II. Surface markers.

Fifteen of 16 lymphoma-derived cell lines and the Faji and P3HR1 cell lines were characterized with regard to certain surface markers, particularly immunoglobulins, complement receptors, Epstein-Barr virus (EBV) receptors, and Fc receptors. Ten lines positive for EBV nuclear antigen (EB-pos) were stained weakly or not at all by antihuman immunoglobulin fluorescein isothiocyanate conjugates, whereas EBV nuclear antigen negative (EB-neg) cell lines stained brightly. EB-pos lines frequently manifested Fc receptors, particularly for 7S antibody, whereas EB-neg lines did not. Receptors for the C3b component of complement and for EBV, which correlated significantly with each other, were expressed to a much lesser extent by EB-neg lines than by EB-pos lines. These findings are pertinent to an understanding of the infrequent association of this virus with American undifferentiated lymphomas of the Burkitt's and non-Burkitt's types.

Antigens, Viral↗

[Establishment of a cisplatin-resistant new human endometrial adenocarcinoma cell line, Sawano cells].

A new human endometrial adenocarcinoma cell line, Sawano cells, was established from an endometrial adenocarcinoma from a 61-year-old woman and has been maintained in vitro for more than 2 years and 10 months. The cells were found to form a monolayer in a mosaic fashion and to tend to pile up. Population doubling time was 43.2 hours at the 10th generation. The modal chromosomal number of the cell fell in a diploid range. Histology of the tumor induced in athymic mice showed it to be a moderately differentiated adenocarcinoma which closely resembled the original human tumor. Estrogen and progesterone receptors were not demonstrated in the in vitro culture cells and in the tumors induced in nude mice, though they were positively demonstrated in the original tumor. The cells had intrinsic cisplatin-resistance (50% inhibition concentration:6.63 micrograms/ml) at 120 hours of contact. We believe this cell line with help us to investigate the biological mechanisms of CDDP resistance.

Adenocarcinoma↗

Use of protamine sulphate for elimination of substrate DNA in poliovaccines produced on continuous cell lines.

Cell substrate DNA was shown to be an abundant contaminant in the clarified preparations of the Sabin type 1, 2 and 3 poliovaccines produced on a continuous cell line (4647). The size of the DNA, as evaluated for the Sabin type 1 poliovaccine, was highly heterogeneous, ranging from 100 to 20,000 base pairs. In view of potential oncogenicity of this DNA a simple and efficient procedure for its elimination is proposed. The method is based on use of protamine sulphate which at the concentration of 2.0 mg ml-1 precipitated cell DNA almost completely without affecting the virus titres.

Animals↗

Genetically engineered pancreatic beta-cell lines for cell therapy of diabetes.

The optimal treatment of insulin-dependent diabetes mellitus (IDDM), which is caused by the autoimmune destruction of pancreatic islet beta cells, would require the regulated delivery of insulin by transplantation of functional beta cells. beta-cell transplantation has so far been restricted by the scarcity of human islet donors. This shortage would be alleviated by the development of differentiated beta-cell lines, which could provide an abundant and well-characterized source of beta cells for transplantation. Using conditional transformation approaches, our laboratory has generated continuous beta-cell lines from transgenic mice. These cells produce insulin amounts comparable to those of normal islets and release insulin in response to physiological stimuli. Cell replication in these beta cells can be tightly controlled both in culture and in vivo, allowing regulation of cell number and cell differentiation. Another challenge to cell therapy of IDDM is the protection of transplanted cells from immunological rejection and recurring autoimmunity. By employing adenovirus genes which downregulate antigen presentation and increase cell resistance to cytokines, beta-cell transplantation across allogeneic barriers was achieved without immunosuppression. In principle, similar beta-cell lines can be derived from isolated human islets using viral vectors to deliver conditionally regulated transforming and immunomodulatory genes into beta cells. The combination of these approaches with immunoisolation devices holds the promise of a widely available cell therapy for treatment of IDDM in the near future.

Animals↗

Protein kinase activity on the cell surface of a macrophage-like cell line, J774.1 cells.

Protein kinase activity was demonstrated on the cell surface of a murine macrophage-like cell line, J774.1 cells, and was characterized in detail. When intact cells were incubated with [gamma-32P]ATP, a transfer of [32P]phosphate into acid-insoluble materials of the cells occurred. This reaction was Mg2+-dependent but cAMP-independent, and Mg2+ could be substituted for by Mn2+. The reaction products were found to be proteins, as revealed by SDS-polyacrylamide gel electrophoresis and autoradiography, with phosphomonester linkages to serine and threonine residues, but not to tyrosine. The results of experiments with chemical and enzymatic treatments as well as Con A-Sepharose column chromatography ruled out the possibility that an acyl-phosphate linkage or phosphomannosylglycopeptide was present in the reaction products. The protein kinase(s) and the reaction products were located on the cell surface of the cells, as shown by the fact that the products were removed by mild trypsinization of cells carefully controlled so that the cells remained in an intact state. Phosphorylation of exogenous proteins (phosvitin and casein) by intact cells further supported the location of the enzyme. The phosphorylated proteins of the cells were found to be metabolically stable and remained on the cell surface even at 120 min after the phosphorylation reaction. Possible roles of ecto-protein kinase activity in macrophage functions and macrophage-activation are also discussed.

Animals↗

Characterization of heparanase from a rat parathyroid cell line.

Cell surface heparan sulfate proteoglycans undergo unique intracellular degradation pathways after they are endocytosed from the cell surface. Heparanase, an endo-beta-glucuronidase capable of cleaving heparan sulfate, has been demonstrated to contribute to the physiological degradation of heparan sulfate proteoglycans and therefore regulation of their biological functions. A rat parathyroid cell line was found to produce heparanase with an optimal activity at neutral and slightly acidic conditions suggesting that the enzyme participates in heparan sulfate proteoglycan metabolism in extralysosomal compartments. To elucidate the detailed properties of the purified enzyme, the substrate specificity against naturally occurring heparan sulfates and chemically modified heparins was studied. Cleavage sites of rat heparanase were present in heparan sulfate chains obtained from a variety of animal organs, but their occurrence was infrequent (average, 1-2 sites per chain) requiring recognition of both undersulfated and sulfated regions of heparan sulfate. On the other hand intact and chemically modified heparins were not cleaved by heparanase. The carbohydrate structure of the newly generated reducing end region of heparan sulfate cleaved by the enzyme was determined, and it represented relatively undersulfated structures. O-Sulfation of heparan sulfate chains also played important roles in substrate recognition, implying that rat parathyroid heparanase acts near the boundary of highly sulfated and undersulfated domains of heparan sulfate proteoglycans. Further elucidation of the roles of heparanase in normal physiological processes would provide an important tool for analyzing the regulation of heparan sulfate-dependent cell functions.

Animals↗

A monovalent cation-sensitive actin-binding factor in a myeloid leukemia cell line.

Cell extracts of a murine leukemia cell line, M1, apparently contain three kinds of actin-gelation factors; a filamin-like protein, and 38K-dimer and 105K-dimer proteins. Unlike gelation by the filamin-like protein, gelation by the latter two proteins is inhibited by low concentrations of KCl. Our study of the 38K protein has been reported elsewhere (Takagi, K. et al., J. Biochem. Tokyo 97, 605-616, 1985). We here describe the purification and characterization of the 105K protein. The 105K protein differs from the alpha-actinin group of proteins in its antigenicity, peptide components and Ca2+-insensitivity. The saturated binding ratio of the protein to purified skeletal muscle actin is 1:8; when this ratio exceeds 1:20, gelation takes place. This gelation is inhibited completely by the presence of 25 mM KCl. Electron microscopy revealed that, in the absence of KCl, the 105K protein/actin mixture forms short actin bundles that are accompanied by a meshwork of short single filaments. The presence of 25 mM KCl did not prevent actin-bundling, but the bundles became longer and the meshwork of short filaments was no longer present.

Actins↗

Expression of calretinin in human mesothelioma cell lines and cell cycle analysis by flow cytometry.

Human mesothelioma cell lines were studied concerning the expression of the calcium-binding protein calretinin (CR), and the relation of the DNA index to their cell cycle. The results obtained for cell lines with different morphological characteristics, were compared to those from human mesothelial cells transfected with SV40 to escape senescence. Immunocytochemical expression of calretinin (confirmed by immunoblot) was observed in all mesothelioma cell lines but not in the control cells. It is suggested that calretinin is active in the first steps of carcinogenesis in all human mesotheliomas and during several stages in the evolution towards malignancy.

Blotting, Western↗

Thy-L antigen expression on rat brain cell lines.

Cell lines derived from the central nervous system of rats were screened serologically for the presence of Thy-1 (theta), a cell surface differentiation antigen shared by brain and thymus of rats and mice. Both cytotoxicity absorption and indirect immunofluorescence assays were performed using a rabbit anti-rat thymocyte serum (ATS) with Thy-1 specificity. The complement-dependent cytotoxicity of ATS detected a mouse-rat cross-reacting determinant of the molecule bearing the Thy-1 antigen. Of 20 lines tested, 1 of 6 neuronal and 7 of 14 non-neuronal lines expressed Thy-1, as judged by their capacity to absorb ATS cytotoxicity for a Thy-1 positive thymoma line. Similar results were obtained in quantitative absorption assays of these lines employing a mouse anti-Thy 1.1 alloantiserum, but the xenoantiserum (ATS) was more sensitive for detecting the rat molecule bearing Thy-1. Indirect immunofluorescence, which was performed on several of the lines, yielded results in complete agreement with the cytotoxicity absorption assays, and revealed a generalized distribution of antigen in a speckled or patchy pattern over the membrane of cell bodies and processes.

Animals↗

Modulation of the plasminogen activator activity of a transformed cell line by cell density.

The effects of variations in cell density on the expression of the plasminogen activator activity of a tumorigenic rat cell line were analyzed. At low cell densities, the plasminogen activator activity per cell was high and independent of cell density. As the cell density increased, the plasminogen activator activity per cell decreased until it eventually became inversely proportional to cell density. Inhibition of the plasminogen activator activity per cell by increases in cell density was not the result of the presence of a soluble inhibitor but seemed to require cell-to-cell contact. The V(max) per cell for the activation of plasminogen changed at high cell densities, but the K(m) did not change. This change in the V(max) per cell was in part the result of a change in the catalytic rate constant for the conversion of plasminogen to plasmin. This was inferred from studies on the kinetics of inhibition of plasminogen activator activity by diisopropyl fluorophosphate as a function of cell density. For cells growing at high densities, the rate of inhibition was constant, exhibiting a second-order rate constant of 2.6 x 10(-2)M(-1) s(-1). For cells growing at low densities, the plasminogen activator activity was inhibited at two different rates, one exhibiting a second-order rate constant of 2.6 x 10(-2)M(-1) s(-1) and the other exhibiting a second-order rate constant of 9.4 x 10(-2)M(-1) s(-1). We discuss the importance of cell density in assays of the plasminogen activator activity of cells, the use of this cell line to study the biochemical basis of the density dependence of plasminogen activator activity, and the density-dependent role of plasminogen activator activity in tumor formation and metastasis.

Animals↗

Tubuloreticular structures in human lymphoid cell lines. A cell biological study.

Human lymphoid cell lines were studied as an experimental model for the spontaneous or induced occurrence of tuburloreticular structures (TRS). It was possible to induce TRS after culturing the EB-3 cell line with 20 mug/ml bromodeoxyuridine (BrUdR) during 96 h. Starvation, culturing at lower temperature (32 degrees) or inhibition of DNA synthesis did not give rise to the production of TRS. The response to BrUdR could be blocked with 60 mug/ml thymidine but not with 60 mug/ml deoxycytidine. The addition of 5 mug/ml cytarabine or the removal of BrUdR at different times resulted in inhibition of TRS induction, indicating that BrUdR had to be incorporated into DNA during at least 48 h. After incorporation, neither the presence of BrUdR nor DNA synthesis was necessary for the production of TRS. These experiments and the finding that in the cell line IHTC-33, which does not produce Epstein-Barr virus associated antigens, TRS were spontaneously present, exclude a correlation between TRS and these antigens. However, the induction of TRS by BrUdR may be related to the activation of another (latent) virus.

Antigens, Viral↗

Control of cell pH in the T84 colon cell line.

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 +/- 0.023 (n = 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 +/- 0.011 pH units/min in the presence of Na+, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na+ independent H+ transport mechanisms in HEPES Ringer. In CO2/HCO3- Ringer, basal cell pH was 7.21 +/- 0.020 (n = 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO2, followed by return to the initial pH at a rate of -0.14 +/- 0.012 (n = 8) pH/min; this return was retarded or abolished in the absence of Cl- or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl-/HCO3- exchange. This exchange was not Na+ dependent. When Na+ was added to cells incubated in 0 Na+ Ringer while blocking Na+/H+ exchange by HMA, cell alkalinization by 0.19 +/- 0.04 (n = 11) pH units was observed, suggesting the presence of Na+/HCO3- cotransport carrying HCO3- into these cells, which was abolished by DIDS. These experiments, thus, show that Na+/H+ and Cl-/HCO3- exchange and Na+/HCO3- cotransport participate in cell pH regulation in T84 cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

Requirement of de novo protein synthesis for aminopterin-induced apoptosis in a mouse myeloma cell line.

Cells synthesize nucleotides through de novo and salvage pathways that require the activities of dihydrofolate reductase (DHFR) and hypoxanthine-guanine phosphoribosyltransfease (HGPRT), respectively. Aminopterin, an inhibitor of dihydrofolate reductase, has been demonstrated to allow HGPRT(-) cells to be negatively selected. However, the pathway by which aminopterin leads to cell death remains to be clarified. In this study, we characterized features of cellular responses induced by aminopterin treatment in P3-X63-Ag8.653, a mouse HGPRT(-) myeloma cell line. Upon treatment with aminopterin, the cells readily underwent an apoptotic process, as assessed by DNA fragmentation assay and electron microscopic analysis. Aminopterin-induced apoptosis was drastically reduced by addition of actinomycin D and cycloheximide, indicating that active RNA and protein synthesis is required for the apoptotic effect of aminopterin. Interestingly, the induction of c-myc gene expression preceded the activity of DNA fragmentation in aminopterin-treated cells. Taken together, these results suggest that cells deficient in the salvage pathway of purine biosynthesis are susceptible to aminopterin-induced apoptosis that requires de novo synthesis of proapoptotic factors, including Myc oncoprotein.

Aminopterin↗

The role of duplication of tumour-derived chromosome 15 carrying the rearranged pvt-1 gene in the transformed phenotype of YACUT T-cell lymphoma x G4 T-cell line somatic cell hybrids in dictating the terminal differentiation program of the parental G4 cell.

Fusion of the YACUT T-cell lymphoma with the Mls-1a-antigen-specific non-tumorigenic T-cell line G4 was previously reported to produce growth-arrested hybrids that could be induced to proliferate in the presence of Mls-1a antigen. The proliferation-suppressed hybrid lines exhibited phenotypic changes as follows: the usually high levels in YACUT of J11d antigen, IL-2 receptor, and c-myb expression, which are markers of immature T cells, were all down-regulated; the G4 T-cell function, i.e., contact helper activity for B-cell proliferation in T/B cell collaboration, was retained. Furthermore, fusion of the YACUT lymphoma with a killer T-cell line produced growth-arrested and tetraploid somatic cell hybrids having killer activity. Thus, in addition to the transformed phenotype (autonomous proliferation in vitro), the antigen-specific non-tumorigenic T-cell line genomes introduced into the YACUT lymphoma suppressed the immature phenotypes of YACUT and imposed their own programming of terminally differentiated traits on the hybrids. Prolonged growth of the proliferation-suppressed hybrid lines by repeated antigenic stimulation was previously reported to result in the appearance of transformed hybrids, which was accompanied by both a reversion of c-myc expression to the levels of YACUT and an increase in the number of chromosome 15. The present study revealed that the amplification of chromosome 15 resulted from the duplication of the tumour-derived chromosome 15 carrying the rearranged pvt-1 gene. However, the differentiated phenotypes of the hybrids remained mostly unchanged upon cell transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗