Showing papers on "Cell culture published in 1968"

Surface IgM-kappa Specificity on a Burkitt Lymphoma Cell In Vivo and in Derived Culture Lines

Abstract: Summary An exceptional Burkitt lymphoma patient, a 16-year-old boy, yielded tumor biopsy cells and derived tissue culture lines that displayed a strong surface accumulation of IgM heavy and kappa light chain specificities judged by direct membrane fluorescence and cytotoxicity tests. This property was maintained unchanged in the course of more than 5 months of serial passage in vitro . A fourth biopsy, obtained from the patient after massive necrosis had been induced in the tumor by cytosine arabinoside chemotherapy, did not show this property at either the biopsy stage or in the derived cell line. The possibility that the neoplastic transformation of lymphoid cells may have afflicted a cell specialized to carry immunoglobulin on its surface may be considered. The phenomenon has to be distinguished from immunoglobulin coating in vivo seen with certain biopsy samples from this and other patients. The latter type of coating can be of IgM, IgG, or IgA nature and disappears rapidly on cultivation in vitro .

Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone.

Abstract: Three clonal strains of epithelial cells were established from a transplantable rat pituitary tumor. These strains have been serially propagated for 14–25 months. They were subcultured every 2–3 weeks. Cells of the original strain have increased by a factor of more than 1040. The generation times of the 3 lines were similar and ranged between 30 and 40 hr. Cells of all 3 strains synthesize growth hormone and secrete it into the culture medium. Growth hormone synthesized in vitro is indistinguishable from normal rat pituitary growth hormone as measured by micro-complement fixation and radioimmunoassay. The specific activity of growth hormone production was estimated to be 20–40 figμng cell nitrogen/24 hr for the most vigorous strain. There has been no decrease in the rate of cell division nor decline in growth hormone secretion since the cells were established in culture. (Endocrinology 82: 342, 1968)

The Establishment of a Cell Line of Human Hormone-synthesizing Trophoblastic Cells in Vitro

Abstract: A human hormone-synthesizing trophoblastic cell system has been established in vitro and may prove to be the first functional human embryonic cell line in continuous culture. Chorionic gonadotropin hormone produced by these cultures serve as a marker for identification of the trophoblastic cell. No interruption in this property nor change in cytologic display has occurred during 1.5 years in continuous culture. The continued proliferation of the undifferentiated cytotrophoblast is being stabilized in serial cultivation.

Cyclic x ray responses in mammalian cells in vitro.

Abstract: Various radiation responses in mammalian cells depend on the position of the cell within its generation cycle (that is, its age) at the time of irradiation. Studies have most often been made by irradiating synchronized populations of cells in vitro. Results in different cell lines are not easy to compare, but an attempt has been made here to point out similarities and differences with regard to cell killing and division delay. In general, survival data obtained so far show that, in cells with a short G1, cells are most sensitive in mitosis and in G2, less sensitive in G1, and least sensitive during the latter part of the S period. In cells with a long G1, in addition to the above, there is usually a resistant phase early in G1 followed by a sensitive stage near its end. (The latter may be as sensitive as mitosis.) Exceptions to the above, especially in some L cell sublines, have been noted, and a possible explanation is given. In Chinese hamster cells, maximum survival after irradiation occurs during S, b...

Transformation of foetal human keukocytes in vitro by filtrates of a human leukaemic cell line containing herpes-like virus.

Abstract: Transformation of foetal human leukocytes followed inoculation with a filtrate of cells of the QIMR-WIL human leukocyte cell line, previously shown to carry a herpes-like virus. Proliferation of leukocytes was generally noted 24–35 days after inoculation, and resulted in the establishment of cell lines resembling those derived from peripheral leukocytes of patients with leukaemia or infectious mononucleosis. The sex of representative transformed lines confirmed that they were of foetal origin. Transformation occurred with two separate filtrates of QIMR-WIL cells, with leukocytes from five individual foetuses, and with leukocytes from bone marrow, thymus and spleen. Various control inocula gave negative results, including culture medium and filtrates of Raji and QIMR-GOR cell lines. Representative transformed lines were found to fix complement with human serum known to react with QIMR-WIL and QIMR-GOR cells; but similar serum showed little evidence of reaction by immunofluorescence. The transformed cell lines studied appeared to be essentially diploid, and subterminal constrictions involving Group C chromosomes were noted in a small proportion of cells in 8/9 lines. In preliminary studies, the transforming factor was found to be sensitive to heat and ether, was sedimented at 70,000 × g and retained by a filter of APD 50 mμ; these results are compatible with the herpes-like virus of the QIMR-WIL cells being the transforming factor.

The Integrated State of Viral DNA in SV40-Transformed Cells

Abstract: It has been shown by a modified DNA-RNA hybridization technique that the nuclei of cells transformed by either polyoma virus or SV40 contain viral DNA.1 This method employs RNA synthesized in vitro with form I viral DNA2–4 and E. coli RNA polymerase. The number of viral DNA equivalents per cell varies from 5 to 60, depending on the cell line. In this communication, we will report on the physical state of the viral DNA in SV3T3 cells, an SV40-transformed cell line that contains 20 SV40 DNA equivalents per cell.

Membranes of animal cells. II. The metabolism and turnover of the surface membrane.

Abstract: Turnover studies of the surface membrane and of cell particulate matter of L cells in tissue culture in logarithmic and plateau phase of growth have been made. The rate of incorporation of isotope into these fractions and the rate of fall of specific activities of labeled L-cell fractions have been observed. The following interpretation of the data appears most likely although other interpretations are possible. Growing and nongrowing cells synthesize approximately similar amounts of surface membrane and particulate material. In the growing cell the material is incorporated with net increases in substance. There is relatively little turnover. In the nongrowing cell newly synthesized material is incorporated, but a corresponding amount of material is eliminated so that there is turnover without net increase of substance. Our results suggest that there is no gross differential turnover between the protein, lipid, and carbohydrate of the surface membrane under the conditions of our experiments. Metabolic inhibitors or omission of amino acids in the culture medium lead to a decrease in synthesis of surface membrane and cell particulates and cause an equivalent decrease in the rate of degradation of surface membrane and of particulates; therefore the synthetic and degradative aspects of turnover appear to be coupled. As cultures of nongrowing cells in suspension or on a glass surface age, their synthetic and turnover capacities diminish. Our results suggest that the cell may exist in a nongrowing state with a level of synthesis similar to that of a growing cell. It can exist in this state with a high level of turnover.

Inhibition of Cell Growth in vitro by Adenosine 3', 5'-Monophosphate

Abstract: Adenosine 3',5'-monophosphate, at a concentration of 40 micrograms per milliliter, inhibits the growth of HeLa and strain L cells in culture. The inhibition becomes progressively greater during the incubation of the cells. Adenosine 5'-monophosphate and adenosine, metabolites of adenosine 3',5'-monophosphate, do not affect the growth of either cell culture. This suggests that 3',5'-monophosphate enters the cell without alteration. Dibutyryl-adenosine 3',5'-monophosphate, reported to have a greater activity than adenosine 3'5'-monophosphate on several tissues, inhibited the growth of the cells much less.

Replication and plaque assay of influenza virus in an established line of canine kidney cells.

Abstract: A plaque assay system has been developed for types A and B influenza viruses in an established line of canine kidney cells (MDCK-USD). In addition to a homogeneous susceptible cell, consistent plaque production depends on the use of highly purified agar (Agarose). This quantitative system was used to determine the rate of adsorption, synthesis, and thermal inactivation of influenza viruses, as well as to determine a dose response curve. Plaque assays on the MDCK-USD line and the parent MDCK line showed that the latter was more sensitive to A/Swine and A(2)/Japan 305 viruses. Titration of standard virus pools in embryonated eggs and MDCK-USD indicated that the cell culture system was as sensitive as the in ovo assay.

Apparent HeLa cell contamination of human heteroploid cell lines

Abstract: INTERSPECIFIC cell culture contamination has been detected several times by karyotypic and immunological procedures1–3. These same measurements are of little value as detectors of intraspecific contamination, but polymorphic variants detectable at the cell culture level can be very useful for this purpose, A survey of twenty heteroploid human cell lines for glucose-6-phosphate de-hydrogenase (G6PD) and phosphoglucomutase (PGM) electrophoretic polymorphisms revealed that all had the same G6PD and PGM phenotypes. These results were interpreted as reflecting widespread contamination of cultures by the first established human cell line HeLa. These findings were presented at the Second Decennial Review Conference on Cell Tissue and Organ Culture4 and have since been extended to include other American Type Culture Collection lines. Further evidence on the stability of these markers in various conditions has been obtained, and in view of the widespread use of cell lines in a variety of investigations it seems important to make these findings readily accessible to other workers.

Chromosome damage induced by plasma of x-rayed patients: an indirect effect of x-ray.

Abstract: Radiation can damage human chromosomes, both in vivo (1-5) and in vitro (6-8). This report describes more fully the results of an experiment devised to distinguish between the direct effects of X-rays on leukocyte chromosomes and a possible indirect effect mediated by way of blood plasma. Blood plasma from patients who had received large doses of X-ray enhanced the degree and frequency of chromosomal aberrations of normal nonirradiated lymphocytes in short-term cell culture (9).Materials and Methods. Six patients with a variety of tumors were chosen because they had received large doses of X-rays through several different ports (see Table I). X-ray integral doses varying from 2.88 to 25.1 × 106 gR were given with a 2 meV van de Graaf generator. Six other patients with similar tumors, studied prior to X-ray, served as controls for the first group. Three of the control patients with tumors were studied before and after X-ray. None of the patients received chemotherapeutic agents or had symptoms of viral inf...

Inducers of interferon and host resistance, V. In vitro studies.

Abstract: Previous reports'-6 from this laboratory recorded that double-stranded ribonucleic acid molecules from numerous sources are efficient inducers of interferon and of resistance to viral infection in vivo and in vitro. The present report describes the induction of resistance against several viruses in a variety of cell cultures by the complex of polyriboinosinic and polyribocytidylic acids (rI:: rC) and presents evidence to associate such resistance with interferon induction. The inactivity of the individual homopolymers, rI and rC, is reaffirmed, and interferoln induction by rI: rC in vitro is shown to be inhibited by exposure of cells to actinomycin D. Materials and Alethods.-(l) Polyriboinosinic acid (rI) and polyribocytidylic acid (rO) were purchased from Miles Laboratories, Elkhart, Indiana. The rI: rC complex was prepared by mixing rI and rC in equimolar coD.eentration in phosphate-buffered salin.e (0.006 M sodium phosphate 0.15 11l NaCl pH 7.0). (2) Cell cultures: The various primary, cell strain, and line cell cultures listed in Table 1 were prepared and maintained by ordinary procedures. T'he RK13 culture is a stable line of rabbit kidnley cells, and the WI-38 and HFL cultures are diploid cell strains of hu:man embryonic lung. The RK13 an-d WI-38 cultures are well documented and the HFL cell strain was developed by Dr. C. Baugh in these laboratories. Rabbit spleen cell suspensions were prepared according to Field et al.2 (3) The viruses used were prepared from the seed stocks of this laboratory. The rhinovirus serotypes were designated according to the number system of Hamparian et al.' (4) Induction of resistance to viral illfection was measured after overnight treatment of cell cultures with rI :rC. Following removal of inducer, interference with virus replication was measured by the plaque-reduction assay.' (5) Interferon induction in primary rabbit kidney cells or in rabbit spleen cell suspensions by rI: rC was assayed by t:ransfer of serial dilutions of the cell supernatant fluids to RK13 cultures in Falcon. flasks. 'rhis was followed by overnight incubation prior to removal and challenge with. vesicular stomatitis virus (VSV) with observation for reduction in plaque formation. RKI.3 cells were used for assay since they were relatively insensitive to iniduction of resistance to VSV by rI: rC, requiring more than 1 ,ug/ml. Hence, these cells were unaffected by the small residual amount of rI:rC in the interferon samples. The interferon titer was the highest dilution of sample which caused at least 50% reduction in plaque formationL. (6) Other pertinent methods are described in the text.

Hemopoietic progenitor cells of W anemic mice studies in vivo and in vitro.

Abstract: The proliferation and differentiation of hemopoietic cells from genetically anemic Wv/Wx,W/Wv, and Wv/Wv mice, and from nonanemic carrier W/+, Wb/+, and Wv/+ mice have been evaluated in vivo by transplantation techniques and in vitro by the agar gel culture method Marrow from anemic and carrier mice contained progenitor cells which were decreased in number and formed small, often rudimentary, colonies in the spleens of irradiated recipient mice Proliferation and differentiation of both erythropoietic and leukopoietic progenitor cells were delayed and reduced, but erythropoiesis was more severely affected than leukopoiesis The severity of the hemopoietic impairment was gene-dose dependent The W gene effect on leukopoietic progenitor cells was not secondary to anemia or to abnormal erythropoiesis The marrow cells of anemic and carrier mice which form colonies of granulocytic and mononuclear cells in vitro were neither decreased in number nor impaired in proliferation and differentiation Hypertransfusion of red blood cells increased the frequency of in vitro colony-forming cells, but not that of in vivo progenitor cells The data demonstrate that colony-forming cells which proliferate in the agar gel cultures in vitro are distinct from the in vivo colony-forming cells and suggest that the former are primitive members of the granulocytic cell line Perhaps in vitro CFU are in an intermediate stage of differentiation between in vivo CFU and myeloblasts, analogous to that which has been suggested for the erythropoietin-sensitive cell in the red cell series W mutant alleles appear to act, therefore, at or very near the beginning of hemopoietic differentiation

"Superinduction" of tyrosine transaminase in hepatoma cell cultures: differential inhibition of synthesis and turnover by actionomycin D.

Abstract: Conitinuous breakdown and replenishment of the macromolecular constituents of animal cells was recognized some time ago in the pioneering experiments of Schoenheimer and his colleagues,' but the significance of degradative processes in regulation of metabolism has become apparenit only recently. Schimke, Sweeney, and Berlin have demonstrated2 that the physiological level of the enzyme tryptophan pyrrolase can be elevated either by stimulation of its synthesis or by inhibition of its degradation, the former process being initiated by glueocorticoid hormones and the latter by the substrate of the enzyme, tryptophan. In. a recent report' from our laboratory, it was shown that degradation of the tyrosine transaminase of liver was blocked when protein synthesis was inhibited by agents such as cycloheximide or puromycin.; under these conditions, the transaminase level was stabilized by conconmitant inhibition. of both synthesis and degradation of the enzyme. At that time it was suggested3 that certain "paradoxical" effects of inhibitors of RNA or protein, synthesis on enzyme levels might result from differential inhibition. of the cellular processes involved in forming an enzyme and of those required for its removal. In the present study, we have analyzed the roles of synthesis and degradation. in the elevation of tyrosine transaminase, which follows the addition of actinomycin D to hormonally induced eell cultures. We find that transaminase synthesis, initially high due to induction by hydrocortisone, is progressively inhibited by the antibiotic. That the transarninase level rises during this interval, despite inhibition of its synthesis, reflects a marked inhibition of degradation of the enzyme. Materials and Methods.-Hepatoma cultures of 'the Reuber H-354 and hepatoma tissue culture (HTC)5 cell lines were grown in monolayer in 250-ml Falcoil plastic flasks conltaining 10 ml of Eagle's basal medium (BME) enriched fourfold with amino acids and vitamins and supplemented with 20% fetal calf serum and 5% calf serum. Penicillin G (100 units/ml) and streptomycin sulfate (100 ,g/ml) were added for routine culturilng. Occasionally, antibiotics have been omitted for several passages and the medium cultured for bacterial contamination. Checks for pleuropuieumonia-like (PPLO) coitatamination also have beein carried out using the method of Barile, Yaguchi, and Eveland6 but with horse serum in place of whole blood in the plating agar. No contaminlation by bacteria or mycoplasmas has been detected. All tissue culture materials were purchased from Grand Island Biological, Co. except penicillin G and streptomycin sulfate, which were obtained from Squibb. During logarithmic growth, both cell lines had a doubling time of approximately 24 hr. Inoculation of cells at a concentration of 1.3 X 105 cells/ml resulted in cultures which enter stationary phase on about day 9. All experilments were performed using 9to 12day (stationary phase) moiiolayer cultures in serum-free IX BME (i.e., unenriched). Reuber H-35 and HTC cells do not grow in serumn-free BME, but will resume growth on readdition of serum after as long as 6 days in its absence. Hydrocortisone (Calbiochem) was dissolved in a minimal volume of ethanol and diluted

Comparative Studies of Fluorinated Pyrimidines with Various Cell Lines

Abstract: Summary The effects of a number of pyrimidine nucleoside analogs on the growth of HeLa, Novikoff hepatoma, and L 5178 leukemia cells in culture have been determined. Information on the biochemical mechanisms of action has been obtained by experiments on the prevention of the inhibition by normal metabolites and by reversal from inhibition by delayed addition of the normal metabolites. In HeLa cells the appropriate normal metabolites prevented the inhibition produced by 5-fluorouracil, 5-fluoro-2′-deoxyuridine (FUDR), 5-fluorouridine, 2′,3′-dehydro-5-fluoro-2′-deoxyuridine, 5-iodo-2′-deoxyuridine, 5-trifluoromethyl-2′-deoxyuridine, and arabinofuranosylcytosine. Delayed addition of the normal metabolites reversed the inhibition produced by the above compounds with the exception of 5-trifluoromethyl-2′-deoxyuridine and 5-iodo-2′-deoxyuridine, which were not reversed by thymidine, and the inhibition by 5-fluorouridine, which was not reversed by uridine, suggesting that the irreversible toxicity was the consequence of incorporation of these analogs into nucleic acids. Cell lines of the Novikoff hepatoma and L 5178Y leukemias were studied that were resistant to FUDR as a result of a loss of thymidine kinase. These cells were effectively inhibited by 2′,3-dehydro-5-fluoro-2′-deoxyuridine, a new analog that is not phosphorylated by thymidine kinase. With the exception of arabinofuranosylcytosine, all the compounds exerted widely differing toxicities to the various cell lines.

Properties and Uses of Human–Mouse Hybrid Cell Lines

Abstract: Staged reduction of the chromosome complement of human–mouse hybrid cells has made possible the identification of the chromosome bearing the human gene for thymidine kinase. Effects of human chromosomes on the growth rate of hybrid cells are examined and the possibility of making further chromosomal assignments for human autosomal genes is discussed.

Studies on the Ontogeny of the Mouse Immune System: II. Immunoglobulin-Producing Cells

Abstract: Summary It has been shown that cells which have the potential to differentiate into immunoglobulin-producing cells appear in the yolk sac, liver and caudal half of the embryo by the 9th day of gestation. Late in pregnancy these cells are found in the thymus, gut, lung, spleen, femur and peripheral blood. Certain of the data suggest that immunoglobulin-producing cell lines and those cells which mediate cell-bound immune responses arise early in gestation as separate cell populations. It has been shown that immunoglobulin synthesis per se is independent of the thymus.

Growth and Morphogenesis of Asparagus Cells cultured in vitro

Abstract: SINCE Steward1 demonstrated the totipotency of plant cells the morphogenesis of plant tissues cultured in vitro has often been investigated. So far, embryoid formation has been found only in dicotyledons. We report here the production of embryoids and their subsequent development into complete plants in a culture of a monocotyledon, Asparagus officinalis L.

Delayed initiation of DNA synthesis in irradiated human diploid cells.

Abstract: MOST studies of the effects of ionizing radiation on the mitotic cycle in mammalian cells have been carried out with rapidly multiplying cells. In vitro systems have included primarily established cell lines or tumour cells. I describe here experiments on morphologically pure cultures of human diploid cells; these cells differ in that they were freshly explanted from normal tissue, and have a very long generation time in vitro. The initiation of DNA synthesis (as measured by thymidine incorporation) appears to be a highly radiosensitive process in these cells, the entry of cells into the synthetic phase being significantly slowed by exposures as low as 10 r. and completely blocked by those over 300 r.

Retention of Differentiated Function in Clonal Animal Cell Lines, Particularly Hormone-Secreting Cultures

Abstract: Clonal hormone-secreting cell lines have been established from animal tumors. These lines include adrenal steroid-secreting cells, growth hormone-secreting cells, steroid-secreting Leydig cells, and ACTH-secreting cells that retain their differentiated function for prolonged periods in continuous culture. In addition, fibroblast cell lines that secrete a material which stimulates steroidogenesis by adrenal cells and Leydig cells are described. A systematic approach to obtain functional cultures and the general problem of retention of differentiated function in cultured cells are discussed.