Showing papers on "Cell culture published in 1970"

Cell and tissue culture

Abstract: Cell and tissue culture , Cell and tissue culture , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Proteolytic Enzymes Initiating Cell Division and Escape from Contact Inhibition of Growth

Abstract: BRIEF treatment with very low concentrations of proteolytic enzymes can bring about a change in the cell surface similar to that occurring in the chemical or viral transformation of normal to malignant cells1. This suggests to us that proteolytic enzymes may not only convert the surface structure into the type seen in malignant cells but that the whole cell may respond to the proteolytic enzyme with initiation of new rounds of cell division and a concomitant escape from contact inhibition of growth, as is the case for malignant cells in tissue culture2.

Topoinhibition and serum requirement of transformed and untransformed cells.

Abstract: The multiplication of tissue culture cells is controlled by several different physiological characteristics, the quantities of which can be measured. Transformed cells are characterized by the values of some of these physiological parameters.

PRODUCTION OF BOTH PROLACTIN AND GROWTH HORMONE BY CLONAL STRAINS OF RAT PITUITARY TUMOR CELLS Differential Effects of Hydrocortisone and Tissue Extracts

Abstract: Several established clonal strains of rat pituitary cells which produce growth hormone in culture have been shown to secrete a second protein hormone, prolactin. Prolactin was measured immunologically in culture medium and within cells by complement fixation. Rates of prolactin production varied from 6.6 to 12 µg/mg cell protein per 24 hr in four different cell strains. In these cultures ratios of production of prolactin to growth hormone varied from 1.0 to 4.1. A fifth clonal strain produced growth hormone but no detectable prolactin. Intracellular prolactin was equivalent to the amount secreted into medium in a period of about 1–2 hr. Both cycloheximide and puromycin suppressed prolactin production by at least 94%. Hydrocortisone (3 x 10-6 M), which stimulated the production of growth hormone 4- to 8-fold in most of the cell strains, reduced the rate of prolactin production to less than 25% of that in control cultures. Conversely, addition of simple acid extracts of several tissues, including hypothalamus, to the medium of all strains increased the rate of production of prolactin six to nine times and decreased growth hormone production by about 50%. We conclude that multifunctional rat pituitary cells in culture show unusual promise for further studies of the control of expression of organ-specific activities in mammalian cells.

Presence of EB virus nucleic acid homology in a "virus-free" line of Burkitt tumour cells.

Abstract: The presence of virus in Burkitt lymphoma cells is hard to confirm, but small amounts of viral DNA have now been revealed by hybridization with DNA from cells of the Raji line of the tumour.

Cytotoxicity and Mode of Action of 5-Azacytidine on L1210 Leukemia

Abstract: This study attempts to determine the primary actions of 5-azacytidine (5-azaCR) on L1210 leukemia. This agent was cytotoxic toward L1210 cells growing in culture with 50 and 90% inhibition dose values of 0.019 and circa 0.15 μg/ml, respectively. 5-AzaCR inhibits the incorporation of tritiated thymidine or deoxyadenosine into DNA to a greater extent than it inhibits the incorporation of tritiated uridine into RNA. Similar results were obtained with ascitic cells isolated from leukemic mice. Equimolar amounts of cytidine reduced the inhibition of DNA synthesis, as well as the inhibition of cell growth in culture caused by 5-azaCR. Uridine, but not deoxycytidine or deoxyuridine, was also effective, but to a lesser extent than was cytidine. With cell-free extracts isolated from L1210 cells in culture, no significant effect was found on enzyme systems directly or indirectly involved in DNA synthesis. With 5-azaCR-4-14C as the precursor, this agent was found to be phosphorylated in all leukemic tissues studied. The majority of phosphorylated products existed as the triphosphate in ascitic and cultured L1210 cells. A portion (10 to 20%) of all these phosphorylated derivatives appeared to be further reduced to deoxyribonucleoside di- and/or triphosphate forms. 5-AzaCR was also incorporated into polynucleotides in all tissues studied and incorporated into both RNA (80 to 90% total incorporated radioactivity) and DNA fractions (10 to 20%) in L1210 cells in culture. In the presence of cytidine, phosphorylation of 5-azaCR, subsequent reduction, and incorporation into polynucleotides were greatly inhibited. A probable mechanism of action of 5-azaCR on L1210 leukemia is proposed.

A Human Tissue Culture Cell Line from a Transitional Cell Tumour of the Urinary Bladder: Growth, Chromosome Pattern and Ultrastructure

Abstract: Cell cultures were made from 18 human bladder tumours. Three cell lines were maintained for seven transfer generations, but all had a "fibroblastic" morphology and a normal diploid karyotype. A fourth line has been maintained for over 80 transfer generations. This was derived from a well differentiated papillary tumour of bladder. Morphologically the light and electron microscopic structure of the cells resembled that of bladder tumours. The cells formed tumour nodules, with a similar structure, when transplanted into hamster cheek pouches. There is a stem line chromosome number of 48. Karyotypes of 60% of the stem line cells had one extra chromosome in Group C and one in Group D.

INTERFERON AND CELL DIVISION, I. INHIBITION OF THE MULTIPLICATION OF MOUSE LEUKEMIA L 1210 CELLS In Vitro BY INTERFERON PREPARATIONS

Abstract: Mouse interferon preparations inhibited the multiplication of mouse leukemia L 1210 cells in stationary suspension cultures. The degree of inhibition was found to correlate with the antiviral titer of the interferon preparations. The factor(s) responsible for inhibition of L 1210 cell multiplication could not be dissociated from interferon by standard physicochemical means.

Cell density-dependent changes of glycolipid concentrations in fibroblasts, and loss of this response in virus-transformed cells.

Abstract: The glycolipids of normal fibroblasts cells at different stages of growth and with differing degrees of susceptibility to contact inhibition have been analyzed, as well as the glycolipids of virally transformed cells. The concentrations (per mg protein) of certain glycolipids, including galactosylgalactosyl-glucosyl-, N-acetylneuraminosylgalactosylglucosyl-, and N-acetylneuraminosyl-(N-acetylneuraminosyl)galactosylglucosyl-c eramide, increase on cell-to-cell contact of susceptible cells. The concentrations of these glycolipids decrease when susceptibility to contact inhibition is lost as a result of extensive passage in vitro or transformation by simian virus 40 or polyoma virus, and in all the malignant cells examined, the concentrations of these glycolipids were independent of cell density.

Selection of a Single Antibody-Forming Cell Clone and Its Propagation in Syngeneic Mice

Abstract: A single antibody-forming cell clone has been selected from primed mice by sequential transfer of limited numbers of spleen cells into irradiated syngeneic mice. The original spleen cell donors had been immunized with dinitrophenylated bovine gamma globulin. Specific antibody molecules in sera of recipient mice were separated by isoelectric focusing on polyacrylamide gels and visualized by 131I-hapten binding and autoradiography. This method provided a marker for antibody-forming cells derived from a single cell clone. This report describes the history of one clone of cells (E9) producing IgG antibody to dinitrophenyl. Clone E9 is long-lived and has been maintained for five transplant generations (over 6 months) by serial transfer of spleen cells into irradiated syngeneic mice. Clone E9 has the following properties: (1) Antibody production strictly depends on antigen, presented either in vivo or in vitro; (2) Induction of E9 anti-dinitrophenyl shows specificity for the carrier protein; (3) Antibody is produced in amounts (2-3 mg/ml serum) comparable with myeloma-protein production by murine plasmacytomas; (4) In the absence of antigen, memory cells have a lifetime exceeding 28 days.

REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : I. Production of Stable, Reversible G1-Arrested Populations in Suspension Culture

Abstract: Suspension cultures of Chinese hamster cells (line CHO) were grown to stationary phase (approximately 8–9 x 105 cells/ml) in F-10 medium. Cells remained viable (95%) for at least 80 hr in stationary phase, and essentially all of the cells were in G1 Upon resuspension or dilution with fresh medium, the cells were induced to resume traverse of the life cycle in in synchrony, and the patterns of DNA synthesis and division were similar to those observed in cultures prepared by mitotic selection. Immediately after dilution, the rates of synthesis of RNA and protein increased threefold. This system provides a simple technique for production of large quantities of highly synchronized cells and may ultimately provide information on the biochemical mechanisms regulating cell-cycle traverse.

Mammalian cell fusion. 3. A HeLa cell inducer of premature chromosome condensation active in cells from a variety of animal species.

Abstract: The phenomenon of premature chromosome condensation (PCC) is induced in unstimulated horse lymphocytes, bovine spermatozoa, Chinese hamster ovary cells, embryonic chick fibroblasts and erythrocytes, Xenopus kidney and mosquito cells by fusing each of these cell types with HeLa cells blocked in mitosis. Thus it becomes possible to visualize chromosomes even from non-multiplying cells of heterologous species, such as, chick erythrocytes and bovine spermatozoa.

Enzymatic block in the synthesis of gangliosides in DNA virus-transformed tumorigenic mouse cell lines.

Abstract: The ganglioside pattern of both SV40- and polyoma virus-transformed mouse cell lines differs from that of the parent cell lines or of cell lines that have transformed spontaneously in tissue culture. This is manifested by a dramatic decrease of gangliosides with an oligosaccharide chain larger than sialyllactose. Present investigations indicate that this change probably cannot be attributed to excessive catabolism of gangliosides, but is caused by impaired synthesis of tri- and tetrahexosyl gangliosides in the virus-transformed cell lines. We present evidence for the block of a required step for the biosynthesis of these ganglioside homologs. The block involves the enzyme catalyzing the transfer of N-acetylgalactosamine from uridine diphosphate N-acetylgalactosamine to hematosides (N-glycolylneuraminyl or N-acetylneuraminylgalactosylglucosyl ceramide). This well-defined enzymatic change opens the way for studies of the biochemical mechanism of the alteration of cell membranes which occurs after transformation by the tumorigenic DNA viruses polyoma and SV40.

Integration of the Deoxyribonucleic Acid of Adenovirus Type 12 into the Deoxyribonucleic Acid of Baby Hamster Kidney Cells

Abstract: In a previous report, evidence was presented that the deoxyribonucleic acid (DNA) of adenovirus type 12 (Ad12) is integrated by covalent linkage into the DNA of baby hamster kidney cells (BHK-21 cells). These studies have been extended. The DNA of Ad12 and that of BHK-21 cells grown in medium containing 5-bromodeoxyuridine could be separated by equilibrium centrifugation in alkaline CsCl density gradients. BHK-21 cells were infected with 3H-labeled Ad12, and the total intracellular DNA was analyzed at various times after infection in alkaline CsCl density gradients. The 3H label in the position of cellular DNA hybridized predominantly with viral DNA and to a lesser extent also with cellular DNA. Replication of viral DNA could not be detected in BHK-21 cells. The appearance of viral 3H label in the density stratum of cellular DNA was not significantly affected when DNA synthesis in Ad12-infected BHK-21 cells was inhibited >96% by cytosine arabinoside. These findings provided additional evidence for integration of Ad12 DNA into the DNA of BHK-21 cells. It could be calculated that 5 to 55 Ad12 DNA equivalents per cell are integrated. Replication of viral or cellular DNA was not required for integration. Inhibition of protein or ribonucleic acid synthesis interfered with integration only slightly.

Evidence for the multiplication of scrapie agent in cell culture.

Abstract: MANY attempts have been made to grow the causative agent of scrapie in cell culture, but there is no unequivocal evidence of multiplication in vitro. Cultures of cells derived from brains of sheep and mice affected with scrapie and from normal animals have been described by Gustafson and Kanitz1. They observed that many of the cells, which appeared to be astrocytes, had enlarged or bizarre nuclei or were multinucleated and these abnormalities were more marked in preparations from animals affected with scrapie. Mice, inoculated with undiluted tissue culture fluids from some of their scrapie preparations, either primary or serially passaged cells, developed the disease. The passaged cells had undergone twenty-three transfers; however, it was not clear whether the scrapie activity observed was derived from the original brains or had been caused by the multiplication of agent to low titre.

The role of immunoglobulins in lymphocyte-mediated cell damage, in vitro. I. Comparison of the effects of target cell specific antibody and normal serum factors on cellular damage by immune and non-immune lymphocytes.

Abstract: Rats were immunized by intraperitoneal injection of target cells (Chang cells). Five days after immunization antibody, lytic to Chang cells in the presence of complement, which separated in Peak I on Sephadex G-150 was detectable. At the same time antibody lytic to Chang cells in the presence of lymphoid cells from unimmunized animals and separating in Peak II on Sephadex G-150 was found. Both antibodies reached a maximum titre between 2 and 4 weeks after immunization. At that time the antibody dependent upon normal lymphoid effector cells was active at more than 1000 times the maximal dilution of complement dependent antibody activity. The Peak II antibody affected both im-immune and non-immune Black Hooded (BH) rat spleen cells in a similar way. Normal rat serum markedly enhanced target cell survival. This effect did not require heat labile components of complement. The enhancing capacity of immune serum appears to be greater than that of normal serum; the reason for this is discussed, but the problem is not resolved.

Changes in Deoxyribonucleic Acid Synthesis Regulation in Chinese Hamster Cells Infected with Simian Virus 40

Abstract: Infection of primary or secondary cultures of Chinese hamster embryo cells with simian virus 40 at a multiplicity of 20 to 50 induced synthesis of the virus-specific intranuclear T antigen in 80 to 90% of the cells within 48 to 72 hr. In the infected cultures, 30 to 50% more cells were recruited into deoxyribonucleic acid (DNA) synthesis than in the controls, whether or not the cultures were confluent. The newly synthesized DNA was mostly cellular, since little virus was produced (as shown by various techniques: immunofluorescence for viral antigen, virus growth curves, and isolation of viral DNA from infected cultures). Transformed cells could be detected a few weeks after infection and produced tumors when inoculated into irradiated animals. Chromosomal changes were observed soon after infection (24 hr). Initially, there was a marked increase in the proportion of polyploid cells (8 to 14%), most of which were chromosomally normal. In a few weeks, a large majority of the infected population was polyploid (30 to 50%). Thus, the polyploid cells have the ability to proliferate. Evidence is presented to suggest that polyploid cells arise by stimulation of cells in the G1, G2, or S phases to undergo two or more successive periods of DNA synthesis without an intervening mitosis. With a subsequent loss or redistribution of chromosomal material, this may lead eventually to a biologically transformed cell; thus, it is suggested that the initial event(s) relevant to transformation occurs at the level of control of cellular DNA synthesis.

Tyrosine transaminase induction by dexamethasone in a new rat liver cell line.

Abstract: A cell line derived from normal, adult rat liver has been established; the cells are similar to hepatocytes, as shown by electron microscopy The addition of dexamethasone to the culture medium induced a three- to sixfold increase in the specific activity of tyrosine alpha-ketoglutarate transaminase; this increase was inhibited by the simultaneous addition of cycloheximide or actinomycin D The latter, when added to cells given prior treatment with dexamethasone, further enhanced the transaminase activity Contact-inhibited cells showed a lower response to dexamethasone than exponentially growing cells

Stable Haploid Cultured Cell Lines from frog Embryos

Abstract: Two haploid cell lines have been established from androgenetic embryos of the frog, Rana pipiens; one line has been maintained in culture for 150 generations, the other for 200 generations Karyotypes of the two lines agree well with the standard for the species although some chromosomes show small differences in length The cells multiply in the same defined basal medium used for culture of other anuran cell lines; this medium consists of the usual amino acids, vitamins, and serum macromolecules plus an exogenous purine source Both the haploids resemble „permanent” cell lines in their prolonged multiplication in culture The two lines differ in their mode of growth, one being epithelial-like, the other forming an overlapping meshwork of fibroblast-like cells Both have the low plating efficiency characteristic of „unaltered” cells These two lines are exceptional in their ability to compete successfully with the diploid variants which arise by endomitosis or cell fusion and which usually overgrow the haploid population The more vigorous line, RPH 682A, should provide the long-desired haploid material for genetic studies in cell culture

Some biochemical aspects of the immune macrophage.

Abstract: Some of the biochemical properties of mouse peritoneal macrophages immune to Corynebacterium ovis were characterised. Total cellular protein of immune cells exceeded that of normal phagocytes by 1·85 times. The activities of 7 hydrolytic enzymes, acid phosphatase, β-glucuronidase, Cathepsin D, lysozyme, BPN-ase, MN esterase and aryl sulphatase were measured in lysed cell suspensions and monolayer cultures. Immune macrophages possessed substantially higher levels of these enzymes than did normal cells. No one enzyme was significantly more associated with the development of cellular immunity than another. Resting immune macrophages consumed significantly less oxygen than normal cells required but were twice as active in glycolysis. ATP levels, in agreement, were 5 times higher in normal macrophages whereas ATP-ase activities were equivalent. Normal macrophages were approximately twice as active in protein synthesis measured by the in vitro incorporation of 14C L-glycine by monolayer cultures than were immune cells. These results were considered in the light of known morphological differences between the 2 cells noted at the ultrastructural level.

Glucocorticoid-induced alteration of the surface membrane of cultured hepatoma cells.

Abstract: Glucocorticoids induce an alteration of the surface of hepatoma tissue culture (HTC) cells as expressed by changes in cell electrophoretic, antigenic, and adhesive properties. The alteration is assayed by the increased adhesiveness of induced cells for a glass surface. The induction process has a lag period of about 3 hr and attains a plateau level after 24–30 hr when 50–80% of the steroid-treated cells are firmly adhered. Less than 10% of untreated cells adhere under the same conditions. Induction is inhibited by actinomycin D and cycloheximide, demonstrates both pH and temperature dependence, and responds to changes in steroid concentration and structure. By contrast, the attachment per se of preinduced cells is not affected by inhibitors of RNA and protein synthesis, fluctuations of temperature and pH, and the presence or absence of the hormone. When the induction process is reversed by removal of steroid or addition of actinomycin D, preinduced adhesiveness is lost with a half-life of 13–24 hr, but in the presence of cycloheximide the loss is accelerated (t1/2 3–5.5 hr). These results suggest that glucocorticoids induce the biosynthesis of a protein which either modifies the cell surface (an enzyme) or is incorporated into surface structures (structural protein).

Differential temperature sensitivity of normal and cancer cells in culture

Abstract: Serially-propagated growing heteroploid and growing diploid cell cultures do not survive incubation at 42° for 24 hours, whereas contact-inhibited diploid monolayers are still viable after at least nine days at this elevated temperature. Heat-treated heteroploid HeLa cells and growing diploid cells exhibit a variety of morphologic abnormalities, but contact-inhibited cells are only minimally affected. A similar differential temperature sensitivity exists in the synthesis of cellular macromolecular components such as DNA, RNA, and protein: incorporation of radioactive precursors is drastically reduced in growing diploid and heteroploid cells after 24 hours at 42°, but not in contact-inhibited cells. Incorporation of labelled glucose, choline, or linolenic acid is actually enhanced in heat-treated contact-inhibited cells.

Macrophage Aggregation in Vitro: A Correlate of Delayed Hypersensitivity

Abstract: A macrophage aggregation test which uses peritoneal exudate cell suspensions (PEC) in tubes in the presence of antigen was found to correlate with cutaneous delayed hypersensitivity. Moreover, the supernatant fluids from lymphoid cell cultures from antigen sensitive animals caused aggregation of nonsensitive PEC in the presence of antigen. Thus, antigen leads to the release of a macrophage aggregation factor (MAF) from sensitive lymphoid cells in culture. By serial dilution, the MAF titers were determined for the supernatant fluids of various lymphoid cell cultures.

The characteristics of animal cells transformed in vitro.

Abstract: Publisher Summary This chapter concerns with the neoplastic transformation of cells in culture. Transformation may occur “spontaneously,” or may be induced by oncogenic viruses and chemical carcinogens. The culture of dissociated animal tissues directly on glass or plastic is established as a simple and valuable technique in many fields of biological research. The methods for the initiation and serial subcultivation of such cultures have remained essentially unchanged since their introduction. During the intervening period, much information has accumulated, although in a rather haphazard fashion, on the growth characteristics of cells derived from various animal species. Some characteristic patterns of behavior have been recognized and cell culture studies have raised many new questions about growth, cell division, metabolism, differentiation, senescence, and transformation. The behavior of cells of different species with regard to their phase of decline and their frequency of spontaneous transformation is represented diagrammatically in the chapter. This representation of behavior of cells from different species is a collation of the results from many published accounts. Some of the studies described in the chapter are related to cells that have spontaneously acquired a high degree of autonomy in vitro. These include BHK2l, NIL-2, and 3T3. These lines retain some of the normal responses to growth controls and lose them when transformed by oncogenic viruses.

Temperature-Dependent Properties of Cells Transformed by a Thermosensitive Mutant of Polyoma Virus

Abstract: In cells transformed by ts-3, a thermosensitive mutant of polyoma virus, the loss of inhibition of DNA synthesis by topographical factors (topo-inhibition), is rendered temperature-dependent, providing evidence that the viral genome controls this essential aspect of transformation. The expression of two other attributes of transformed cells, growth in agar and serum requirement for initiation of DNA synthesis in a wound in culture, is not made temperature-dependent. In productive infection of BALB-3T3 cells by ts-3, virus-induced stimulation of cellular DNA synthesis and movement is rendered temperature-dependent.