Prevention of Malignant Change in Mammalian Cells During Prolonged Culture In Vitro

Mixed cultures of epithelial cells and fibroblasts, derived from primary cultures of the skin of embryo rats, grown always in rubber-stoppered T-60 flasks, first yielded a transplantable tumor from the 52nd passage, at the end of 13 months of frequently repeated subculture. A group of subcultures, derived from the 22nd passage, grown under the same medium, with the addition of 1% oxyhemoglobin, failed to yield a tumor in 23 months of repeated subculture. A return of these cultures to the regular medium with oxyhemoglobin, yielded a tumor in 4 months, after 12 more passages. Cultures of transformed cells that had regularly yielded a transplantable tumor, for 6 years, up to the 305th passage, continued to yield transplantable tumors when 1% oxyhemoglobin was added to the medium. The cells remained highly atypical, microscopically, and there was no indication of reversal of the malignancy. Although oxyhemoglobin in the medium of cell cultures seems to have had the ability to keep malignancy in abeyance, it did not reverse the established malignant transformation of the cells.     

Effect of Double-Stranded Viral RNA on Mammalian Cells in Culture

During bovine enterovirus infection of Ehrlich ascites tumor cells, large amounts of double-stranded RNA accumulate. Addition of this double-stranded RNA to uninfected cells leads to rapid cell death. This is not a result of infectious virus production. Neither single-stranded RNA nor heat-denatured double-stranded RNA has this effect. Similar experiments with synthetic double-stranded polymers, poly(I).poly(C) and poly(A).poly(U), show that they are only slightly toxic at the concentrations used. The effect of the double-stranded RNA is nonspecific for cells of different origins. The implications of this finding in relation to the cytopathic effects of picornavirus and to cancer chemotherapy are discussed.     

Characterization of Developing Adult Mammalian Erythroid Cells Separated by Velocity Sedimentation

By virtue of their different sedimentation rates at unit gravity, different classes of erythroblast have been separated from the bone marrow of the anaemic rabbit. Each class of erythroblast has been characterized by morphological and biochemical criteria. The classes thus characterized form a series of cell types leading from the large immature erythroblast to the marrow reticulocyte. The fractionation procedure permits complete separation between erythroblasts which synthesize RNA and those which are no longer active in RNA synthesis.     

Release of Pyrophosphate by Normal Mammalian Articular Hyaline and Fibrocartilage in Organ Culture

Calcium pyrophosphate dihydrate crystals are found most frequently in fibrocartilaginous tissue and to a lesser extent in hyaline articular cartilage. Previous investigators found that pyrophosphate (PPi) was released into the medium by immature rabbit hyaline cartilage and osteoarthritic human cartilage in organ culture but not by normal human or mature rabbit cartilage. By employing a sensitive fluorometric assay for PPi and correcting for hydrolysis of PPi during the incubations, we detected PPi release by all normal mammalian cartilage studied. PPi release per mg wet weight of lapine and canine cartilage was paralleled by uronic acid production. Meniscal fibrocartilage, the most common site of calcium pyrophosphate deposits, also elaborated PPi.     

Preservation of Normal Behavior by Enucleated Cells in Culture

BHK-21 fibroblasts and BSC-1 epithelial cells were enucleated with cytochalasin B. The enucleated cells were trypsinized and replated; their subsequent behavior was monitored by light optical techniques. The results demonstrate that the information necessary for attachment, spreading, shape formation, pinocytosis, contact inhibition, and cell locomotion is present in enucleated cytoplasm.     

Effect of Near-Ultraviolet and Visible Light on Mammalian Cells in Culture II. Formation of Toxic Photoproducts in Tissue Culture Medium by Blacklight

Near-ultraviolet radiation was found to be lethal for mammalian cells in Dulbecco's modified Eagle's medium without serum or phenol red. Irradiation of the cells with near-ultraviolet light while the cells were in phosphate-buffered-saline abolished the lethal effect. When only the medium was irradiated followed by the addition of unirradiated cells and serum, the cells were still killed. The photoactive components of the medium for this effect were riboflavin, tryptophan, and tyrosine. When riboflavin was deleted from the medium being irradiated and added later, almost no killing was detected. Irradiation of salt solution of riboflavin and tryptophan or riboflavin and tyrosine, resulted in cell killing. Little or no killing resulted when riboflavin, tryptophan, or tyrosine was irradiated singly.The formation of photoproducts toxic for mammalian cells appears to involve photodynamic action. Experiments utilizing Dulbecco's or similar media without proper controls may produce anomalous results from light illuminating the laboratory.     

Altered Enzymes in Drug-Resistant Variants of Mammalian Tissue Culture Cells

Two selective procedures are compared in an effort to isolate variants of mouse L cells containing structural gene mutations. Among the resulting variant cloned cell lines are found two types of alterations in the enzyme hypoxanthine phosphoribosyl transferase (EC 2.4.2.8) (1): enzyme with altered kinetic constants causing in vivo and in vitro resistance to 8-azaguanine; and (2) enzyme with altered heat sensitivity in vitro. These results support the view that tissue culture cell variants can arise from structural gene mutations.     

Selective Degradation of Abnormal Proteins in Mammalian Tissue Culture Cells

The degradation rates of several missense mutants of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) in mouse L cells are compared to those of the wild-type enzyme. Although the rates of total protein breakdown in the mutant cell lines are identical to that of the parental L cell line, defective molecules of hypoxanthine-guanine phosphoribosyltransferase present in the mutant cell lines are degraded much faster than the wild-type enzyme. The level of defective phosphoribosyltransferase molecules present in the mutant cell lines is inversely proportional to the breakdown rate. This observation indicates that the major factor determining the concentrations of the defective phosphoribosyltransferases is their specific degradation rate. These results strongly support the hypothesis that abnormal proteins are selectively degraded in mammalian cells.     

The Effect of an Effortful Swallow on the Normal Adult Esophagus

The effect of an effortful swallow on the healthy adult esophagus was investigated using concurrent oral and esophageal manometry (water perfusion system) on ten normal adults (5 males and 5 females, 20-35 years old) while swallowing 5-ml boluses of water. The effects of gender, swallow condition (effortful versus noneffortful swallows), and sensor site within the oral cavity, esophageal body, and lower esophageal sphincter (LES) were examined relative to amplitude, duration, and velocity of esophageal body contractions, LES residual pressure, and LES relaxation duration. The results of this study provide novel evidence that an effortful oropharyngeal swallow has an effect on the esophageal phase of swallowing. Specifically, effortful swallowing resulted in significantly increased peristaltic amplitudes within the distal smooth muscle region of the esophagus, without affecting the more proximal regions containing striated muscle fibers. The findings pertaining to the LES are inconclusive and require further exploration using methods that permit more reliable measurements of LES function. The results of this study hold tremendous clinical potential for esophageal disorders that result in abnormally low peristaltic pressures in the distal esophageal body, such as achalasia, scleroderma, and ineffective esophageal motility. However, additional studies are necessary to both replicate and extend the present findings, preferably using a solid-state manometric system in conjunction with bolus flow testing on both normal and disordered populations, to fully characterize the effects of an effortful swallow on the esophagus.     

Effect of Insulin on Sterol and Fatty Acid Synthesis and Hydroxymethylglutaryl CoA Reductase Activity in Mammalian Cells Grown in Culture

The effects of insulin on the synthesis of sterols and fatty acids and on the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), a rate-limiting enzyme for sterol synthesis, were studied in mammalian cells grown in culture. While in some established cell lines sterol synthesis was not affected significantly by the hormone, in the nonpermanent human and animal cells the synthesis of lipids, especially that of sterols, as well as the activity of the reductase were stimulated following an incubation with insulin in a medium containing serum albumin for a few hours or longer. These effects of insulin were also demonstrable in the presence of solvent-extracted serum, which itself increases sterol synthesis and reductase activity. In medium containing whole serum insulin was ineffective. Addition of glucose decreased sterol synthesis as well as reductase activity. The effects of insulin were prevented by cycloheximide and are probably due to an increased synthesis of 3-hydroxy-3-methylglutaryl CoA reductase or of a protein that regulates its activity.     

Membrane Changes and Adenosine Triphosphate Content in Normal and Malignant Transformed Cells

Transformed fibroblasts had a low content of ATP when grown at a high cell density and a high content of ATP when grown at a low cell density. Concanavalin A agglutinated transformed cells with a low, but not those with a high, ATP content. Transformed cells with a high ATP content gained agglutinability after ATP depletion by inhibitors of the energy-generating systems, and those with a low ATP content lost their agglutinability after restoration of a high ATP content by glucose. Fixation of the surface membrane by formaldehyde, glutaraldehyde, or LaCl(3), inhibited agglutination of cells with an ATP content that allows agglutination. Normal fibroblasts grown at a high or a low cell density were not agglutinated by concanavalin A. Depletion of the cellular ATP content of normal cells induced agglutination only in cells grown at a high, but not at a low, cell density. A similar number of concanavalin A molecules was bound to the surface membrane of agglutinating and nonagglutinating fibroblasts. It is suggested that a high content of ATP inhibits the movement of concanavalin A binding sites, and that a low content of ATP allows, in transformed cells, a new distribution of binding sites to form the clusters required for cell agglutination. Agglutinability of transformed cells is determined by ATP content, and in normal cells changes in the content of ATP are by themselves not sufficient to induce agglutination. Transformed cells, therefore, do not have a control, presumably for membrane stability, that exists in normal cells.     

Appearance in Trypsinized Normal Cells of Reactivity with Antibody Presumably Specific for Malignant Cells

Trypsinization of normal human diploid cells (WI-38 and MRC 5) resulted in the appearance of complement-fixing reactivity with an immunoglobulin (anti-HeLa G globulin), prepared against a purified HeLa (malignant human) cell antigen (G), which reacts with various malignant human cell lines and tumors but not with certain normal human cells. The presence of receptors in the nonreacting, untrypsinized normal human cells and the specificity of the reactive groups that appeared after trypsinization was established by the fact that the antibody could be completely absorbed with large quantities of packed, untrypsinized human cells but not with similar quantities of either rabbit or guinea-pig kidney tissue-culture cells, which did not react with this antibody either before or after treatment with trypsin. The change produced by trypsinization is thus similar to the previously demonstrated appearance of reactive groups with the same anti-HeLa G globulin in normal human cells at certain times after infection with herpes simplex and vaccinia viruses.The fact that the trypsinized WI-38 cells absorbed more antibody than the same number of cells before trypsinization indicated that trypsinization resulted not only in the appearance of reactivity with antibody but also in a greater concentration of combining receptors, which is unlike the situation with lectins producing agglutinability without an increase in the number of receptors. Moreover, the fact that absorption with trypsinized normal cells removed larger amounts not only of the antibody reacting with the trypsin-treated WI-38 cells but also of antibody that reacts with WI-38 cells infected with herpes simplex virus and with the malignant HEp-2 cells, suggests that the combining groups that emerge after trypsinization of the normal human cells are the same or similar to those present in malignant human cells (HEp 2) and to those that emerge after infection of the normal human cells with herpes simplex virus.     

Colony Formation by Normal and Leukemic Human Marrow Cells in Culture: Effect of Conditioned Medium From Human Leukocytes

"Conditioned medium" obtained fromcultures of human peripheral leukocytespromoted the growth of human marrowcells in cell culture. This material alsopermitted the growth of small coloniesfrom the marrow of patients with acutemyelogenous leukemia in relapse; in itsabsence, only occasional colonies wereobserved.

Submitted on June 30, 1970 Accepted on July 24, 1970     

Dynamics of Metabolism of Normal and Virus-Transformed Chick Cells in Culture

Application of steady-state tracer technique to normal and transformed cells in tissue culture allows quantitation of intracellular pool sizes of many metabolites and determination of rate of carbon flow along diverse paths. Using a unique apparatus to control the environmental conditions, we show that the glucose carbon flow into tricarboxylic acid cycle intermediates and amino acids is unchanged upon transformation. The increased glycogen formation and glycolysis varies with the glucose concentration in the medium, correlates with the faster glucose transport of transformed cells, and cannot be explained by a difference in growth rate alone.     

The Effect of Phytohaemagglutinin on Human Foetal Cells Grown in Culture

Cell suspensions of foetal thymus, liver, spleen and bone marrow have been grown in cell culture and the effect of adding PHA observed. ³H thymidine uptake by the cells after exposure for a measured time was quantitated by autoradiographic counts performed on the initial suspensions and following culture. The initial unstimulated liver and thymic cell suspensions had a high level of ³H thymidine uptake, while that of the spleen and bone marrow cells was low. After 3 days culture the unstimulated cells from the liver, thymus and bone marrow showed very little ³H thymidine uptake, while that of the spleen was slightly increased. Foetal thymic and splenic cells responded to PHA while liver and bone marrow cells did not. The findings suggest that foetal cells of similar morphology may have different origins in the reticulo-endothelial system.     

Effect of Hemin on the Synthesis of Hemoglobin and Other Proteins in Mammalian Cells

The initiation of globin synthesis in intact reticulocytes and in reticulocyte lysates is maintained by the addition of hemin. The specificity of this effect has been studied to determine whether it is restricted to hemoglobin and erythroid cells. In intact reticulocytes, hemin (500 muM) enhances the synthesis of carbonic anhydrase as well as of hemoglobin. Similar enhancement of protein synthesis is observed on addition of hemin (500 muM) to intact Krebs II ascites tumor cells, in cell-free extracts of these cells, added hemin (50 muM) increases endogenous protein synthesis and the translation of exogenous rabbit globin messenger RNA. These results provide evidence that the effect of hemin is not restricted to globin, and they suggest that hemin may enhance protein synthesis in at least some nonerythroid cells.     

Conventional Treatment and its Effect on Survival of Malignant Pleural Mesothelioma in Western Australia

Abstract: Conventional treatment and its effect on survival of malignant pleural mesothelioma in Western Australia. A. W. Musk and S. D. Woodward, Aust. N.Z. J. Med., 1982, 12 , pp. 229–232.
Analysis of the survival of all 81 cases of pathologically confirmed malignant pleural mesothelioma in Western Australia between January 1957 and December 1980 has revealed a median survival from diagnosis of 5-1 months (mean 7–8 months). The average time between presentation and diagnosis was 3–4 months. Survival was better in younger subjects and subjects selected for surgery but was unrelated to sex, symptoms at onset, history of asbestos exposure, tumour morphology, therapy other than surgery or year of presentation. The selection of subjects at earlier clinical stages for surgical intervention is considered to account for their longer survival. The outlook for patients with this disease remains poor and there is still no optimism for future advances in therapy.     

Replacement of Nerve-Growth Factor by Ganglionic Non-Neuronal Cells for the Survival In Vitro of Dissociated Ganglionic Neurons

Nerve-growth factor is known to cause a considerable increase in the number of neurons putting out processes and surviving in cell cultures of dissociated dorsal-root and sympathetic ganglia from embryonic chicks. Similar effects of nerve-growth factor have now been noted with cultures of dissociated dorsal-root ganglia from newborn mice or rats. In all three sensory ganglionic systems, the effects of the nerve-growth factor on fiber production and neuronal survival could be mimicked, in the absence of the factor, by adequate increase of the non-neuronal cells in the cultures. The results suggest a hypothesis that views the role of the nerve-growth factor as subordinate to that of the non-neuronal cells.