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Spontaneous human mononuclear cell cytotoxicity to cultured tumor cells: reproducibility of serial measurements with the use of a chromium-51-release microcytotoxicity assay.

Twenty-three healthy human adults with naturally occurring (spontaneous) mononuclear cell cytotoxicity (MCC) to cultured human tumor cells were evaluated in a serial study over 23 months to determine whether their initial MCC levels remained stable. A 3-hour 51Cr-release microcytotoxicity assay, in which the HBT-3 cancer-derived cell line with HeLa markers and the ALAB breast cancer-derived cell line were used, was standardized and its suitability for such serial studies was evaluated. The data showed that the 51Cr-release assay is reproducible and suitable for use in serial studies of MCC. The intrinsic technical error for a single assay averaged 12.5% (2-26%) coefficient of variation. The greatest sources of technical variation with time were the passage level of the target cells and the temperature at which the effector cells were stored and handled. When these technical factors were controlled, naturally occurring MCC was fairly reproducible with time. Of the healthy individuals with initial high or low MCC levels, 61% tended to retain these levels in a stable fashion, but the remaining 39% studied showed fluctuations of MCC greater than 33% coefficient of variation.

Blood Preservation

Characterization of macrophage chemotaxins in tumor cell cultures and comparison with lymphocyte-derived chemotactic factors.

Culture fluids from five murine sarcomas were chemotactic for syngeneic peritoneal macrophages in vitro. Peritoneal macrophages from mice infected with Mycobacterium bovis, strain Bacillus Calmette-Guérin, were more responsive to the chemotactic factor in tumor cultures than were normal macrophages. Peritoneal granulocytes, however, did not significantly respond to this factor. The level of chemotactic activity in tumor cultures paralleled cell growth for all five tumors; maximal levels occurred during log growth. Culture medium alone or fluids from proliferating spleen cell cultures stimulated with mitogens did not have detectable chemotactic activity. Chromatography of the tumor culture fluids resulted in a single peak of chemotactic activity in the 15,000-molecular weight range on Sephadex G-100 and at about 7.5 mmho/cm specific conductance on diethylaminoethyl cellulose. By both biological and physicochemical characteristics, the chemotactic activity in tumor culture fluids was different from mouse lymphocyte-derived chemotactic factor.

Animals

Effects of tumor cell culture supernatants on macrophages.

It is proved that supernatants of various tumor cell cultures, as well as cell-free ascitic fluid and sera from tumor bearing animals induce the detachment of macrophages from glass, the loss of their ability to form pseudopodia, the inhibition of their migration and the permeability alteration of their membranes. The cytotoxic effect is not produced by normal tissues, excepting placenta. The effect of tumor cell culture supernatants depends on the characteristic evolution time of each tumor; irradiation diminishes this effect. The involvement of macrophages in immune surveillance and response makes the results interesting for the tumor-host immune relation.

Animals

Characterization of mouse mammary tumor viruses from primary tumor cell cultures. II. Biochemical and biophysical studies.

Primary mammary tumor cultures of RIII, GR, DD, BALB/c, and BALB/cfC3H mice were examined for mouse mammary tumor virus (MuMTV) production. Levels of production of 12-32 mug virus protein/day/75-cm2 culture flask could be maintained for 30-50 days with daily virus harvests. The viruses from tumor cell cultures of these mouse strains contained DNA polymerase with a strong preference for Mg++ over Mn++ as the divalent cation, a characteristic of DNA polymerase of MuMTV from mouse milk. These viruses from tumor cell cultures were excellent sources of MuMTV 3H-complementary DNA (complexed to 60-70S RNA) and radioactive 60-70S RNA, sufficiently free of contaminating murine leukemia virus nucleic acids, that can be used in molecular hybridization experiments. The effects of several culture parameters on MuMTV production were also studied.

Cells, Cultured

Mixed lymphocyte--tumor cell culture reaction in cancer patients.

To measure immunological reactivity or recognition of tumor antigens, mixed lymphocyte--tumor cell culture reaction (MLTR) was studied using a micromethod. The peripheral lymphocytes of cancer patients were mixed in vitro with autochthonous tumor cells that had been irradiated with 60Co at a dose of 6,000 rad just before use. The lymphocyte blastogenic response was estimated by the incorporation of 3H-thymidine in the acid-insoluble fraction and expressed as counts per minute. The stimulation index equal to or greater than 2.0 was arbitrarily expressed as positive blastogenic response in the present experiment. Positive blastogenic responses to autochthonous tumor cells were observed in 10 of 34 patients with various solid tumors. The intensity of blastogenic response to tumor cells was correlated with that of the non-specific responses to PHA (r=0.4732, P less than 0.01). These results indicated that non-self tumor-associated antigens, which could induce in vitro blastogenic response of autologous lymphocytes, were present in some part of human tumors.

Adult

Cultivation of the erythrocytic stages of Plasmodium berghei in Leydig cell tumor cultures.

Twelve different established cell-lines were used in attempts to cultivate the erythrocytic stages of Plasmodium berghei, P. vinckei vinckei, P. coatneyi or P. knowlesi. Intracellular parasites were seen in only mouse Leydig cell testicular tumor (LCT) cultures inoculated with red cells infected with P. berghei. Intracellular parasites were present at 15 to 96 h after inoculation, being most numerous at 36 h. Most intracellular stages were rings, trophozoites, schizonts and merozoites; gametocytes were few in number and present only at 36 and 48 h. Intracellular parasites were normal in general morphology and staining characteristics at 15 to 48 h, but were abnormal after 72 h. Infected host cells exhibited progressive nuclear and cytoplasmic degenerative changes, which ultimately resulted in death of the cell. Uninfected cells appeared normal. The ability of parasites in LCT cultures to produce infections upon injection into mice was similar to that obtained with control cultures without LCT cells.

Animals

Glycoproteins from human colonic adenocarcinoma. Isolation and characterization of cell surface carcinoembryonic antigen from a cultured tumor cell line.

Alterations in cell surface glycoproteins have been implicated in malignancy. We examined surface membrane proteins of a cultured cell line, SKCO-1, which had been derived from a human colonic adenocarcinoma. Cell surface labeling of SKCO-1 cells with galactose oxidase, followed by reduction with sodium borotritide, revealed five major labeled glycoproteins upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. At least three additional labeled glycoproteins could be detected if galactose oxidase treatment was preceded by neuraminidase treatment. Some, but not all, of the glycoproteins could be iodinated by lactoperoxidase. The predominantly labeled glycoprotein (GPI) had a molecular weight of 200,000 and co-migrated in SDS gel with carcinoembryonic antigen (CEA). GPI was not removed from the cell surface by EDTA, hypertonic saline, or sonication but was released from the membrane by detergents. This glycoprotein was subsequently purified using lectin-agarose columns and gel filtration. GPI was judged homogenous by protein- and carbohydrate-stained SDS-polyacrylamide gels and had an amino acid composition similar to that of CEA. The carbohydrate composition of GPI was qualitatively similar to CEA but quantitatively distinct. GPI had a greater proportion of sialic acid and galactosamine and less fucose and glucosamine than CEA. Immunological studies, however, demonstrated identity between GPI and CEA. A study of the turnover rate of GPI showed it to have a half-life of 5 days.

Adenocarcinoma

Affinity isolation of cultured tumor cells by means of drugs and hormones covalently bound to glass and Sepharose beads.

Isoproterenol, corticotropin (ACTH), and triodothyronine immobilized on glass and Sepharose beads by diazotization procedures have been shown to interact with cultured tumor cells of "target tissue" origin. Cells used were rat glioma cells (C6), rat adrenal tumor cells (Y-1), and rat pituitary tumor cells (GH3). The rat glioma cells bound principally to immobilized isoproterenol, whereas the rat adrenal tumor cells bound to immobilized corticotropin, and rat pituitary tumor cells bound to immobilized triiodothyronine. Binding was inhibited by preincubation of the cells in soluble drug or hormone. With C6 cells there was a positive correlation between adenylate cyclase [ATP pyrophosphate-lyase (cyclizing, EC 4.6.1.1] stimulation and the degree of binding to the immobilized isoproterenol. Norepinephrine, bound through the ethanolamine side chain via an amide linkage, did not bind cells, demonstrating specific structural requirements for drug-cell interactions. HeLa cells were shown to bind tightly to diphtheria toxin coupled to Sepharose beads via an amide bond. This binding was inhibited by prior incubation of the Sepharose toxin with purified antitoxin. Toxin bound to Sepharose via an azo bond did not bind cells. These data suggest that the cell affinities are due to cell surface receptors interacting with the immobilized drugs and hormones, and that the observed affinities possibly reflect the relative receptor complement of these cells.

Adrenal Gland Neoplasms

Unexpectedly low incorporation of isotopic acetate into lipids of Ehrlich ascites tumor cells cultured in lipid-poor medium.

The present communication reports data on the lipid biosynthesis of Ehrlich ascites tumor cells grown in culture media supplemented with modified sera. Whereas the metabolisms of [14C]pyruvate and [14C]mevalonate are identical in all media tested, the incorporation of [14C] acetate is higher in medium with dialyzed serum than in medium with delipidized serum; it is suppressed in the absence of all lipids in culture medium. Cellular integrity is not impaired in modified media. The results indicate that acetyl-CoA synthetase of Ehrlich ascites tumor cells is not regulated by exogenous lipids as is known to be the case in nonmalignant cells.

Acetate-CoA Ligase

Steroidogenesis and extracellular cAMP accumulation in adrenal tumor cell cultures.

ACTH stimulated steroidogenesis and cAMP (adenosine 3',5'-monophosphate) accumulation in an adrenocortical mouse tumor cell line (clone Y1) with Kd values which differed by more than one order of magnitude (5.2 X 10(-11) M and 7 X 10(-10) M, respectively). All of the cAMP formed in response to added ACTH appeared extracellularly in 5- or 30-min incubations. ACTH, at 5 and 10 muU/ml, stimulated steroidogenesis to 25% and 40% of maximum activity; and increased the extracellular accumulation of cAMP 1.4-fold and 2.3-fold, respectively. The effects of ACTH appeared to be via an action on intracellular ATP, specific for cAMP and dependent on an ACTH-sensitive adenylate cyclase system. These observations indicate that ACTH increases cAMP accumulation in Y1 cells at virtually all steroidogenic concentrations and suggest that cAMP is an essential component of ACTH-stimulated steroidogenesis.

Adenosine Diphosphate

Anomalous reactions of mouse alloantisera with cultured tumor cells. I. Demonstration of widespread occurrence using reference typing sera.

M0use alloantisera produced against different specificities of the K, I, and D regions of the H-2 gene complex reacted as immunogenetically anticipated with normal lymphoid target cells of different haplotypes in cytotoxicity and indirect immunofluorescence tests. These same alloantisera, however, produced anomalous positive reactions when tested on cultured MCA-induced sarcoma cells from B10 background H-2 congenic mice. Absorption experiments demonstrated that the anomalous activity in these sera was directed against a tumor membrane antigen(s) which was distinct from H-2 region specificities against which the reference alloantisera were produced, and which was shared in common by multiple cultured sarcoma lines. Similar anti-tumor antibody activity could be demonstrated in the serum of older (greater than 12 weeks) but not younger normal unimmunized mice of the strains used as recipients for alloantiserum production. It is suggested that the observed anamalous anti-tumor activity in these alloantisera may be due to the presence of antibodies reactive with envelope antigens of murine leukemia virus which are expressed on sarcoma cells maintained in culture.

Animals

Dihydrofolate reductase in primary brain tumors, cell cultures of central nervous system origin, and normal brain during fetal and neonatal growth.

Dihydrofolate reductase (DHFR) was measured during the development in rats of brain tumors induced following inoculation with avian sarcoma virus. Increasing activity of this enzyme in brain was correlated with the course of primary brain tumor growth. The specific activities of DHFR in primary human brain tumor tissues were comparable to those found in avian sarcoma virus-induced brain tumors in rats. Specific activities of DHFR in cell cultures derived from human and rat primary intracranial gliomas and sarcomas were up to 6 times those found in adult rat liver. The presence of DHFR in neoplasms of central nervous system origin is relevant to the development of folate antagonists which, unlike methotrexate, can readily cross the blood-brain barrier. In normal developing rat brain, DHFR specific activity was high in embryos at 19 days of gestation and declined thereafter, until at 20 days after birth the activity was very low. The methotrexate titration assay was used to measure enzyme levels in the brains of fetal and newborn rats, and good correlation with the spectrophotometric assay was observed. The pattern was different in liver, showing maximum activity 11 days after birth and retaining high activity in adult liver. Both the cofactor requirement and the sensitivity to methotrexate indicate that the enzyme in the brain is DHFR.

Animals

Anomalous reactions of mouse alloantisera with cultured tumor cells. II. Cytotoxicity is caused by antibodies to leukemia viruses.

Certain alloantisera prepared in mice against H-2 region membrane antigens were found to be unexpectedly cytotoxic for murine sarcoma and leukemia cells in culture. This anomalous cytotoxicity was shown to be the result of antibody in these alloantisera directed against the p15 and gp70 envelope proteins of Mu LV which were present on the surface of the tumor target cells. Sera from aged unimmunized mice of strains used for the preparation of alloantisera also contained antibodies against MuLV protein p15 and gp70 that were cytotoxic for sarcoma and leukemia cells, which indicates that these antibodies occurred naturally in mice. These results independently confirm earlier findings of the widespread occurrence in mouse serum of antibodies reactive with MuLV. The presence of antibody against MuLV in mouse serum which can cause cytotoxic reactions with tumor cells points to the fact that particular caution should be used during the typing of murine sarcomas or leukemias for cell surface antigens, since mouse antisera may yield cytotoxicity (or other serologic reactions) based on anti-MuLV specificities, rather than on anticipated antigens.

Animals

Potentiation of the cytotoxic action of melphalan and "activated" cyclophosphamide against cultured tumor cells by centrophenoxine.

Centrophenoxine, without antitumor activity itself, enhanced the cytotoxic action of melphalan and "activated" cyclophosphamide against mouse P388 lymphoma and rat W256 carcinosarcoma cells growing in static suspension culture. The concentration of alkylating agent required for 99% cell-kill was approximately halved when centrophenoxine was also present during exposure to the antitumor drug. Maximum potentiation by centrophenoxine of the cytotoxic action of melphalan occurred when cells were exposed to the two agents simultaneously; little or no potentiation was observed when cells were exposed to centrophenoxine before or after exposure to the alkylating agent.

Animals