Showing papers on "Cell culture published in 1971"

Murine Leukemia Virus: High-Frequency Activation in vitro by 5-Iododeoxyuridine and 5-Bromodeoxyuridine

Abstract: Cells of embryos of the high leukemic mouse strain AKR can be grown in culture as virus-negative cell lines However, these lines and clonal sublines uniformly have the capacity to initiate synthesis of murine leukemia virus Exposure of the cells to 5-iododeoxyuridine or 5-bromodeoxyuridine induced synthesis of virus in as high as 01 to 05 percent of the cells; many of the cells were producing virus as soon as 3 days after initiation of treatment Induction of virus by these drugs is several orders of magnitude greater than that obtained with any other treatment tested These studies indicate that the full genome of murine leukemia virus is present in an unexpressed form in all AKR cells and provide a potentially powerful technique for activating leukemia virus genomes in other cell systems

The role of three cytoplasmic fibers in BHK-21 cell motility. I. Microtubules and the effects of colchicine.

Abstract: Microtubule breakdown in the presence of 5 or 40 µg/ml of colchicine is observed in BHK-21/C13 fibroblast-like cells Several morphological and physiological effects are noted in the absence of microtubules: (a) the cells transform from fibroblast-like to epithelial-like cells; (b) the normal pattern of intracellular birefringence changes and a juxtanuclear cap of birefringent filaments is formed; (c) time-lapse cinematography demonstrates that cell locomotion is inhibited in colchicine-treated cells, even though membrane ruffling persists The results are discussed in terms of the specific roles of microtubules in cultured cell motility and possible functional relationships of the three types of cytoplasmic fibers seen in BHK-21 cells

Restoration of Several Morphological Characteristics of Normal Fibroblasts in Sarcoma Cells Treated with Adenosine-3':5'-Cyclic Monophosphate and Its Derivatives

Abstract: Sarcoma cells growing in tissue culture have morphological and growth characteristics different than normal fibroblasts. Several of the morphological characteristics of normal fibroblasts are regained when the cells are incubated with dibutyryl-cyclic AMP or butyryl-cyclic AMP (0.1-1 mM), or cyclic AMP (3 mM) plus theophylline (1 mM), but not with ATP, ADP, AMP, adenine, or adenosine (1-7 mM). The cell bodies become elongated; distinct narrow processes are formed. With prolonged incubation, the cells show less tendency to pile up or become polygonal. Further, L-929 and Rous sarcomatransformed hamster cells orient in parallel arrays characteristic of contact inhibition. The cells retain their altered morphology as long as the butyryl-cyclic AMP is present, but revert after its removal. Experiments with cycloheximide, puromycin, and actinomycin D indicate that protein Synthesis, but not RNA synthesis, is required for the response. Microtubular proteins may be involved. No response is observed with normal fibroblasts or with various epithelial cells. The data suggest that cyclic AMP may be an important factor in the determination of morphology of normal fibroblasts and this function may be lost or altered during transformation.

Induction of Murine C-Type Viruses from Clonal Lines of Virus-Free BALB/3T3 Cells

Abstract: Each clone of BALB/c mouse embryo cells that has been tested can be induced to form C-type virus. The individual cells therefore contain a complete copy of the genetic information for making the murine RNA tumor viruses.

Rapid Cell Culture Assay Technique for Murine Leukaemia Viruses

Abstract: INFECTION of susceptible mouse cells in culture with murine leukaemia viruses (MLV) does not cause any observable change in cellular morphology, even though continuous virus replication in these cells can often be demonstrated. Complement-fixation1 and fluorescent antibody2 techniques as well as interference3 or potentiation4 of focus formation by “defective” murine sarcoma viruses have all been used successfully to detect and to quantitate in vitro infection of mouse cell cultures with MLV. These techniques, however, are less than ideal because they involve special reagents and lengthy incubation, or because they are relatively insensitive, Klement et al.5 have shown that the XC cell line6, derived from a rat tumour induced by the Prague strain of Rous sarcoma virus, undergoes syncytium formation when present in mixed cultures together with MLV-infected mouse cells. This phenomenon of mixed culture cytopathogenicity has been used to develop a plaque assay for murine leukaemia viruses in mouse cell cultures (unpublished observations of W. P. Rowe et al.).

The Analysis of Malignancy by Cell Fusion: I. Hybrids Between Tumour Cells and L Cell Derivatives

Abstract: A wide range of different kinds of malignant cell were fused with certain derivatives of the L cell line and the ability of the resulting hybrid cells to grow progressively in vivo was examined. In all cases the highly malignant character of the tumour cells was suppressed by fusion with the L cell derivatives, whether or not these had metabolic defects that facilitated selection of the hybrid cells. So long as the hybrid cells retained the complete chromosome complements of the two parent cells, their ability to grow progressively in vivo was very limited, for tumours composed of such unreduced hybrids were not found. However, when they lost certain specific, but as yet unidentified, chromosomes, the hybrid cells regained the ability to grow progressively in vivo and gave rise to a tumour. These findings thus indicated that the L cell derivatives contributed something to the hybrid that suppressed the malignancy of the tumour cell, and that this contribution was lost when certain specific chromosomes were eliminated.

I. hybrids between tumour cells and l cell derivatives

Abstract: SUMMARY A wide range of different kinds of malignant cell were fused with certain derivatives of the L cell line and the ability of the resulting hybrid cells to grow progressively in vivo was examined. In all cases the highly malignant character of the tumour cells was suppressed by fusion with the L cell derivatives, whether or not these had metabolic defects that facilitated selection of the hybrid cells. So long as the hybrid cells retained the complete chromosome complements of the two parent cells, their ability to grow progressively in vivo was very limited, for tumours composed of such unreduced hybrids were not found. However, when they lost certain specific, but as yet unidentified, chromosomes, the hybrid cells regained the ability to grow progressively in vivo and gave rise to a tumour. These findings thus indicated that the L cell derivatives contributed something to the hybrid that suppressed the malignancy of the tumour cell, and that this contribution was lost when certain specific chromosomes were eliminated.

pH as a Determinant of Cellular Growth and Contact Inhibition

Abstract: 1) Both the growth rate and the maximum population density of several normal, virus-transformed, and cancer cells were markedly pH-dependent; the optimum varied from pH 6.9 to 7.8. At the optimum pH, some diploid human cells attained population densities comparable to those of cancer or virus-transformed cells. Contact inhibition of growth is facilitated by repeated fluctuations of pH in nonphysiological ranges, and may not be an intrinsic and necessary attribute of diploid cells in culture. 2) At pH 8.3, at which there was little or no cellular multiplication, the protein content per cell increased 2- to 5-fold over a period of 10-16 days, and was slowly reversed to normal concentrations on restoration of pH to the optimal range. 3) Uridine uptake by contact-inhibited human cell cultures was stimulated by refeeding with salt solution, and to the same extent as by complete (serum-supplemented) growth medium; that immediate increase did not involve the reinitiation of cellular growth and multiplication. Contact inhibition was, however, reversed in 2-4 days by an appropriate increase in the serum concentration of the medium.

Cultivation in vitro of cells derived from a human osteosarcoma

Abstract: A cell line was derived from an osteosarcoma. Cells grew to high population density in liquid medium and formed colonies in agar medium. The cell line consisted of polygonal or fusiform cells resembling the cells of the original tumor. No collagen fibers or calcium apatite microcrystals could be demonstrated in the cultured cells by electron microscopy nor were virus particles detected. Isoenzyme studies of the cell line revealed the “B” band of glucose-6-phosphate dehydrogenase. Chromosome studies of the cell line revealed a range of stem line number from 58 to 65; chromosome counts ranged from 47 to 183. Some cells had marker chromosome of varied morphology and some had minute chromosomes. Chromosome breaks were found in 30.5% of the cells.

Characterization of the factor in L-cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture.

Abstract: Medium harvested from cultures of mouse L-cells contains “conditioning factor activity” (CFA) that may be detected by its ability to stimulate colony formation by mouse marrow cells in culture. The active material has been characterized and appears to be a glycoprotein with properties similar to those reported for the colony-stimulating activity in human urine. Characterization of trypsin-digested material indicated that only part of the molecule is essential for biological activity. The CFA has been partially purified from serum-free L-cell conditioned medium, and evidence has been obtained that radioactivity derived from labelled valine or glucosamine may be incorporated into the factor. L-cell conditioned medium appears to be a convenient source of partially-purified, highly active material, suitable for use in studies on its mechanism of action.

Priming: a nonantiviral function of interferon.

Abstract: No interferon is made by L cells when they are infected with MM virus. However, several thousand units of interferon are produced when interferon-treated L cells are infected with MM virus. We call the conversion of cells, from nonproducers to producers, priming. The time required for cells to become fully primed is dependent on the interferon concentration with which they are incubated. Primed cells produced interferon earlier than normal cells stimulated by other inducers. Cells which were exposed to interferon in the presence of inhibitors of protein synthesis became fully primed yet developed no virus resistance. Also, primed cells produced interferon in response to low concentrations of polyriboinosinic acid · polyribocytidylic acid that did not induce interferon in normal cells. Therefore, priming appears to be a function of interferon separable from its antiviral activity. Several other picornaviruses that failed to induce interferon in L cells, human embryonic lung cells, or monkey kidney cells did induce interferon when these cells had been primed by homologous interferons.

Further Changes in Differentiation State Accompanying the Conversion of Chinese Hamster Cells to Fibroblastic Form by Dibutyryl Adenosine Cyclic 3′:5′-Monophosphate and Hormones

Abstract: The morphological conversion in vitro of Chinese hamster ovary cells to a fibroblast form by a relatively large amount of dibutyryl adenosine cyclic 3′:5′-monophosphate, or by a combination of small amounts of this compound and testosterone, is attended by appearance of the following additional properties: acquisition of strict contact inhibition of growth; reorientation of the random growth pattern into one in which cells grow parallel to their long dimension; disappearance of the randomly distributed, knob-like, pseudopodal structures around the cell periphery; induction of collagen synthesis; and decrease in the ability to be agglutinated and rounded up by plant agglutinins and specific cell antibodies. The changes in these characteristics are consistent with the conversion from a malignant to a normal fibroblastic state. This conversion is under genetic control, as demonstrated by the production of specific mutants with altered characteristics. The response to testosterone is specific since steroids like estradiol and hydrocortisone are inactive, and others have limited activity. Some prostaglandins are equal in activity to testosterone and 5α-dihydrotestosterone. This system appears useful in study of the regulation of phenotypic expression in mammalian cells.

Isoleucine-mediated regulation of genome repliction in various mammalian cell lines.

Abstract: Summary When suspension cultures of Chinese hamster cells (line CHO) are grown in isoleucine-deficient F-10 medium supplemented with dialyzed sera, the entire population of cells accumulates in a state of G1 arrest within 24 to 36 hr. Merely by the adding back of isoleucine, the entire population initiates DNA synthesis and subsequently divides in synchrony. This phenomenon is not readily observed in cells infected with pleuropneumonia-like organisms. Cultures of mouse L and Syrian hamster BHK21 cells also accumulate in G1 under conditions in which isoleucine is specifically depleted from the culture medium, and, upon addition of isoleucine, synthesis of DNA and resumption of cell-cycle traverse commence in synchrony. These results suggest that isoleucine-mediated regulation of genome replication may be a generalized phenomenon in mammalian cells. Speculations are presented regarding possible roles for isoleucine (or its derivatives) in initiation of genome replication.

Enhancement of Phagocytosis by Interferon-Containing Preparations

Abstract: Exposure of mononuclear cells from the mouse peritoneal cavity to interferon (IF)-containing mouse sera enhanced phagocytosis of colloidal carbon particles by the cells. The same effect was observed when the cells were exposed to IF-containing cell culture harvest free of serum. The magnitude of this effect of IF-containing preparations paralleled the titer of IF and was not related to the dilution of various IF-containing serum specimens tested. The factor responsible for the enhancing effect was stable at pH 2, inactivated by trypsin, and nonsedimentable at 105,000 × g. Heating at 60 C for 1 hr destroyed it, and its kinetics of heat inactivation paralleled that of the antiviral activity of IF. A period of incubation of phagocytic cells with IF-containing serum was necessary before a maximum level of enhancement was reached, and once established was not removable by repeated washing of cells. The kinetics of the production of the enhancing factor in mice injected with Newcastle disease virus was essentially identical to that of the simultaneous production of IF as measured by antiviral activity. Contrary to the effect of mouse IF preparations, human IF preparation did not enhance the activity of mouse phagocytes. It appears, therefore, that the phagocytosis-enhancing factor falls within the present definition of IF.

Regulation of Adenosine 3′:5′-Cyclic Monophosphate Concentration in Cultured Human Astrocytoma Cells by Catecholamines and Histamine

Abstract: Norepinephrine, epinephrine, and histamine cause a rapid increase in the concentration of adenosine 3′:5′-cyclic monophosphate (cAMP) in a tumor astrocyte cell line derived from a primary culture of a human glioblastoma multiforme. The catecholamine-induced increase in cAMP is dependent on the cell density, being far greater in cells in the log phase of growth than in cells near terminal density. The response to norepinephrine is inhibited 50% by 0.01 μM propranolol, a blocking agent of β-adrenergic receptors. In contrast, the effect of histamine on cAMP concentration varies only slightly from log-phase growth to terminal density, and is not inhibited by 10 μM propranolol. The results suggest that astrocytoma cells have independent receptors for catecholamines and histamine. Further, if the astrocytoma cell is an adequate model of the normal glial cell, these results suggest that astrocytes in human cerebral cortex may be sensitive to norepinephrine and histamine.

C-type virus particles in pig kidney cell lines.

Abstract: Pig kidney cells of the PS or PK2a line have proved useful for the study of group B arboviruses (Westaway, 1966; de Madrid & Porterfield, 1969). While following the course of arbovirus replication in PS cells by thin section electron microscopy, it became clear that the PS cell line was carrying a non-cytopathic virus morphologically indistinguishable from the oncogenic C-type virus particles of murine leukaemias (Bernhard, 1960; Dalton, 1962). The PS cell line is known to be chronically infected with swine fever virus (Shimizu et al. 1969), and to avoid this possible complication, attention was next turned to another pig kidney cell line, PK 15, which is known to be free from swine-fever infection. Numerous C-type particles were found in thin sections prepared from control PK 15 cells. To exclude the possibility that both PS and PK 15 cells had become infected, possibly with a murine virus, during handling in this Institute, a fresh supply of PK 15 cells was obtained from the American Type Culture Collection (designation CCL 33, passage 133).

Expression of differentiated functions in hepatoma cell hybrids: reappearance of tyrosine aminotransferase inducibility after the loss of chromosomes.

Abstract: Hybrids from a cross of rat hepatoma cells with diploid epithelial cells from rat liver have been studied with respect to karyotype and expression of two functions limited to the hepatoma parent: high level of the enzyme tyrosine aminotransferase (EC 2.6.1.5; L-tyrosine:2-oxoglutarate aminotransferase) and its inducibility with steroid hormones. The hybrids that contain the complete chromosomal complements from both parents show low enzyme activity and no inducibility. One hybrid clone, and all of its derivatives, which have lost 30-40% of the chromosomes initially present, show enzyme inducibility. Induction of tyrosine aminotransferase in the hepatoma and hybrid cells responds similarly to inhibition by cycloheximide and actinomycin D, and to steroid concentration. The enzymes from induced and noninduced hepatoma cells and from induced hybrid cells are similar in heat sensitivity and intracellular distribution; those from noninduced hybrid and diploid rat epithelial cells are different.

Biochemical Differentiation in Reaggregating Brain Cell Culture

Abstract: Dissociated cells from embryonic mouse brain reassociate in rotation culture to form aggregates. During cell culture the specific activities of choline acetyl-transferase (EC 2.3.1.6), acetylcholinesterase (EC 3.1.1.7), and glutamate decarboxylase (EC 4.1.1.15) in the aggregates increase up to twenty-fold, a phenomenon that approximates some of the biochemical events in the development of the mouse brain.

pH AND POPULATION DENSITY IN THE REGULATION OF ANIMAL CELL MULTIPLICATION

Abstract: Sparse and dense cultures of chick embryo cells were affected differently by pH. The rates of cell multiplication and of thymidine-3H incorporation into DNA of dense cultures were increased as the pH was increased from 6.6 to 7.6. At pH higher than 7.6 the rate of multiplication decreased slightly in the dense cultures, but the rate of thymidine-3H incorporation continued to increase. The discrepancy was due in part to cell death and detachment at very high pH, and in part to a more rapid uptake of thymidine-3H at very high pH. Sparse cultures were much less sensitive to pH reduction and, when a suitably conditioned medium was used to minimize cell damage, very sparse cultures grew almost as well at pH 6.7 as at higher pH. The rates of cell multiplication and thymidine-3H incorporation at low pH decreased in the initially sparse cultures before they reached confluent cell densities. There was no microscope evidence of direct contact between plasma membranes of cells at these densities although the parallel orientation indicated that the cells were influencing locally each other's behavior. Even at much higher cell densities, electron microscopy revealed large intercellular gaps partly filled with a fragmentary electron-opaque material suspected to be glycoprotein. Wounding experiments showed that pH affected cell migration in a manner similar to its effects on cell multiplication. Low pH inhibited cell migration, but those cells which migrated into the denuded region multiplied as rapidly at low pH as at high pH. The effects of pH on growth were correlated with effects on the uptake of 2-deoxyglucose-3H. Dense populations of cells inhibited by low pH were stimulated to incorporate thymidine-3H by the addition of small amounts of diethylaminoethyl-dextran. Rous sarcoma cells at high cell density were less sensitive to pH than were normal cells at the same density, but were more sensitive than sparse normal cultures. The results suggest that cell growth is inhibited through the combined effects of both lowered pH and high cell density on cell surface permeability.

Morphological, oncogenic, and karyological characteristics of Syrian hamster embryo cells transformed in vitro by carcinogenic polycyclic hydrocarbons.

Abstract: Summary Quantitative transformation was induced by carcinogenic polycyclic hydrocarbons in cells plated to produce discrete colonies; transformation was not seen in parallel studies in which cells were treated with pyrene or solvent only. Transformation was defined as a criss-crossing of cells that produced altered colonies not seen in controls. The correlation of the morphologically altered colonies with tumor production was determined by establishing cell lines from altered and normal colonies and analyzing them in terms of morphology, oncogenicity, and karyology. The patterns of various cell lines, when produced by the same carcinogen, differed in types of cells present as well as in degree of altered growth pattern. In cloning experiments, colonies formed which usually had more than one type of morphological transformation. Cells of established lines derived from each transformed culture formed tumors when tested by s.c. inoculation into hamsters. No tumors were produced when control cells were inoculated into irradiated animals. The transformed cell lines analyzed for karyological changes had a low incidence of chromosomal markers. The cell lines, regardless of carcinogen used for transformation, had chromosome modes classified as hypodiploid and hyperdiploid; the tumor-derived cultures were quite similar to the transformed cell line in chromosome mode except for two which had shifted to the hypotetraploid range. The lack of common karyotypic changes demonstrates that the alterations were trivial and represent a random occurrence which is independent of the transformation. Since the transformed colonies were transformed only with carcinogens and were responsible for cell lines which produced tumors when injected into hamsters, whereas control untransformed cell lines failed to produce tumors, it is concluded than an in vitro model is reliable for studies of carcinogenesis and that in vitro studies are relevant to in vivo cancers.

Expression of differentiated functions in hepatoma cell hybrids. I. Tyrosine aminotransferase in hepatoma-fibroblast hybrids.

Abstract: The inducible enzyme tyrosine aminotransferase (TAT) has been investigated in hybrids between rat hepatoma cells and mouse fibroblasts. In the latter the TAT baseline activity is low, and in the presence of steroids does not change. By contrast, the hepatoma cells have high TAT activity, and this activity increases by a factor of 4-6 in the presence of steroids. The hybrid cells, like the fibroblasts, have low TAT activity and are not inducible. Heat inactivation curves demonstrate that the hybrid cells contain both parental forms of TAT, and therefore contain the parental genes specifying the enzyme. The presence in the hybrids of detectable rat TAT and the total absence of its inducibility suggest that a second gene is involved in the regulation of TAT inducibility, and that this gene is not expressed in the hybrids.

Contact-inhibited revertant cell lines isolated from SV40-transformed cells. I. Biologic, virologic, and chemical properties.

Abstract: Two contact-inhibited "revertant" cell lines were isolated from an SV40-transformed mouse 3T3 cell line (SV-3T3) after exposure to 5-fluoro-2'-deoxyuridine. Revertant cells resembled 3T3 cells morphologically and grew to saturation densities which were similar to those of 3T3 cells; however, revertant cells readily formed both single and multinucleated giant cells in confluent cultures. SV40 virus was rescued from revertant cells by fusion with permissive monkey cells. The rescued virus transformed 3T3 cells with the same efficiency as wild type virus, and produced transformed colonies which were phenotypically similar to those produced by wild type virus. The revertant cells also resembled normal 3T3 cells in that they contained higher quantities of sialic acid than SV-3T3 cells. An inverse correlation was found between the saturation density of cells and their sialic acid content. Collagen content, however, of revertant cells was similar to that of SV-3T3 cells. The data presented suggest that the property of contact inhibition in revertant cells is related to the sialic acid content of the plasma membrane and that changes in sialic acid content of transformed cells are not directly specified by the viral genome.

Spontaneous and virus induced transformation in cell culture.

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Inability of UV-Irradiated Lymphocytes to Stimulate Allogeneic Cells in Mixed Lymphocyte Culture

Abstract: Cells exposed to UV light are not able to induce DNA synthesis in allogeneic cells. HL-A typing before and after irradiation gives identical results. It is suggested that active cell metabolism and no

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS II. Ultrastructural Study

Abstract: The ultrastructural appearances of normal 3T3, SV40-transformed 3T3 (SV-3T3), and F1A revertant cell lines are compared. Both confluent and subconfluent cultures are described after in situ embedding of the cells for electron microscopy. There is striking nuclear pleomorphism in F1A revertant cells, with many cells having large nuclei compared to the less variable nuclear morphology of both normal 3T3 and SV-3T3 cells. Under the culture conditions used, deep infoldings of the nuclear envelope are prominent in growing cells, e.g., subconfluent normal 3T3 and confluent SV-3T3 cells. Such infoldings are infrequently seen in cultures which display contact inhibition of growth, e.g., normal 3T3 or F1A revertant cells grown just to confluence. In confluent cultures, the cytoplasmic organelles in revertant cells closely resemble those of normal 3T3 cells. In both normal and revertant cells in confluent culture, the peripheral cytoplasm (ectoplasm) has many 70 A filaments (alpha filaments), which are frequently aggregated into bundles. Alpha filaments are also abundant in the ectoplasm near regions of cell-to-cell apposition and in the motile cell processes (filopodia). The abundance and state of aggregation of alpha filaments correlates with contact inhibition of movement and growth in these cell lines since fewer bundles of alpha filaments are seen in growing cells than in contact-inhibited cells. This observation suggests that these filaments may be an important secondary component in the regulation of contact inhibition of movement and, possibly, of growth in normal and revertant cells.