ABSTRACT
Antitumor ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); edelfosine) induces apoptosis in cancer cells, sparing normal cells. We have found that the apoptotic action of ET-18-OCH(3) required drug uptake and Fas in the target cell. Failure to accomplish one of these requirements prevents cell killing by the ether lipid. In human lymphoid leukemic cells, ET-18-OCH(3) does not promote Fas or FasL expression and ET-18-OCH(3)-induced apoptosis is not inhibited by pre-incubation with an anti-Fas blocking antibody that abrogates cell killing mediated by Fas/FasL interactions. ET-18-OCH(3)-resistant normal human Fas-positive fibroblasts do not incorporate ET-18-OCH(3), but undergo apoptosis upon ET-18-OCH(3) microinjection. Murine fibroblasts L929 and L929-Fas, stably transfected with human Fas cDNA, do not incorporate ET-18-OCH(3) and are resistant to its action when added exogenously. Microinjection of ET-18-OCH(3) induces apoptosis in L929-Fas cells, but not in wild-type L929 cells. Confocal laser scanning microscopy shows that ET-18-OCH(3) induces Fas clustering and capping during triggering of ET-18-OCH(3)-induced apoptosis. Microinjection-induced apoptosis and Fas clustering are specific for the molecular structure of ET-18-OCH(3). Our data indicate that ET-18-OCH(3) induces apoptosis via Fas after the ether lipid is inside the cell, and this Fas activation is independent of the interaction of Fas with its natural ligand FasL. This explains the selective action of ET-18-OCH(3) on tumors since only cancer cells incorporate sufficient amounts of the drug.
Subject(s)
Antineoplastic Agents/toxicity , Apoptosis/physiology , Membrane Glycoproteins/physiology , Phospholipid Ethers/toxicity , fas Receptor/physiology , Animals , Antineoplastic Agents/pharmacokinetics , Apoptosis/drug effects , Biological Transport , DNA Fragmentation , Fas Ligand Protein , HL-60 Cells , Humans , Jurkat Cells , K562 Cells , L Cells , Mice , Microinjections , Models, Biological , Phospholipid Ethers/administration & dosage , Phospholipid Ethers/pharmacokinetics , Signal Transduction , Tumor Cells, CulturedABSTRACT
Filming of cultured HeLa cells using time-lapse cinevideomicrography techniques, with exposure to an extremely low frequency electromagnetic field allowed the direct observation of a localized cellular destruction process caused by a white light-electromagnetic field interaction. This phenomenon was not observed with normal human fibroblasts.
Subject(s)
Electromagnetic Fields , Electromagnetic Phenomena , Cell Count , Cell Cycle , Cell Survival , Fibroblasts , HeLa Cells/pathology , Humans , LightABSTRACT
The effect of an electromagnetic field of 100 Hz during 5 msec on HeLa, MMT and LMMB cell lines in vitro was investigated by means of videophotomicrography. An inhibition of cell growth of cancer cells was observed after the simultaneous action of light and electromagnetic field. The combined effect was also proved using plating efficiency tests.
Subject(s)
Electromagnetic Fields , Electromagnetic Phenomena , Fibroblasts/cytology , HeLa Cells/pathology , Mammary Neoplasms, Experimental/pathology , Animals , Cell Division , Cell Line , Humans , Mice , Video Recording/methodsABSTRACT
This work deals with the effect of thiazolidine-4-carboxylic acid (thioproline) upon several in vitro established human cell lines. Times-lapse microcinematography is used to study the action of thioproline on HeLa cells, employing high doses during a short time, and continuous exposure to medium and small doses. The effect of thioproline in mixed cultures of HeLa cells and human lymphoblasts is described. Special attention is paid to the observed cell cycle phase specificity, and to the phenomenon described as reverse transformation that has been associated to the drug. Marked differences is the sensitivity to thioproline are observed for the different types of in vitro established cell lines. The effect is always produced during the G1 and S phases of the cell cycle. Cell changes are produced only after contacting with one another, and the biochemical reactions which are proper of cell contact inhibition of growth and/or genotypic reverse transformation were never observed, so that the mechanism of action of the drug has to be revised. From a morphodynamic point of view, thioproline is an interesting drug because it produces peculiar effects which differ markedly from those produced by the other cancer chemotherapy agents. Thioproline could prove to be useful for cancer chemotherapy if used in combination with other drugs, knowing that it is a low activity phase specific antineoplastic drug and not a drug producing reverse transformation.
Subject(s)
Antineoplastic Agents/toxicity , Microfilming , Thiazoles/toxicity , Cell Division/drug effects , Cell Line , Cell Survival/drug effects , Contact Inhibition/drug effects , HeLa Cells , Humans , KB Cells , Microfilming/methods , ThiazolidinesABSTRACT
Cell respiration (CR) and glycolysis (GL) are the main sources cell energy, since along their metabolic pathways ATP is produced. Expressed as microM/100 mg/h, normal cells produce 63 by CR, 0.2 by aerobic GL, and 9.37 by anaerobic GL, while cancer cells produce 35 by CR, 18 by aerobic GL, and 29 by anaerobic GL. The ascites fluid from EAC increases the anaerobic GL to 38, while it does not change the aerobic GL to 7 and diminishes the CR to 26. Insulin produces a lowering of CR to 26, aerobic GL to 26 and anaerobic GL to 22. Glucose inhibits CR and stimulates GL. Ribose does not modify CR and inhibits GL. Mannose inhibits both CR and GL. Ribonuclease increases GL in the presence of glucose but not of ribose. Glucose-phosphate and ribose-phosphate have no action because they do not enter into the cell. Expressed as QLN2/100 mg, the main localization of GL is the cytosol (480), but it is significant in the nucleus (170), and diminishes in microsomes (100) and mitochondria (52). Mitochondria inhibit the cytosol glycolytic activity when they are either in the usual proportion they have in the cell or in a higher proportion. It is curious the observation that a diminution of the relative concentration of mitochondria with regard to cytosol (1/100 to 1/1000) produces a marked increase of GL. The addition of nuclear fraction stabilizes the cytosol-mitochondria complex and modifies the metabolic pathway of the CO2 that is produced during the GL.
Subject(s)
Carcinoma, Ehrlich Tumor/metabolism , Energy Metabolism , Glycolysis , Animals , Glycolysis/drug effects , In Vitro Techniques , Insulin/pharmacology , Mice , Mitochondria/drug effects , Mitochondria/metabolismABSTRACT
A modification of the antiblastogram, a technique originally used for predictive anti-cancer drug testing, is employed in this work to select microorganisms producing antineoplastic antibiotics.
Subject(s)
Antibiotics, Antineoplastic/biosynthesis , Bacteria/metabolism , Bacteriological Techniques , HeLa Cells , HumansABSTRACT
The technique of the antiblastogram is described. The antiblastogram is used for the in vitro study of anti-cancer chemotherapy agents and predictive drug testing.
Subject(s)
Antineoplastic Agents/pharmacology , Animals , Cell Line , Drug Evaluation, Preclinical/methods , Humans , MiceABSTRACT
Several biological and biochemical techniques are combined to get the isolation of tumor-specific and tumor-associated antigens (TSA and TAA). Specific antisera to plasma membrane TSA and TAA from the mouse Ehrlich ascites cancer (EAC) cells are induced by selective immunization of immunologic chimera rabbits to normal mouse antigens. The specific anti-cancer immunosera are absorbed with normal antigens to remove any possible residual normal antigens. The antibodies to TSA and TAA are purified by several cycles of discontinuous ascending chromatography in dextran gel particles, and used as a ligand for affinity chromatography in cyanide bromide-activated agarose gel. The material offered to the column consists of EAC cell plasma membrane fragments solubilized by ultrasonic waves, and isolated by discontinuous sucrose density gradient centrifugation. The marked cytotoxicity inhibition of antisera to EAC cells obtained by the TSA and TAA eluted from the affinity chromatography column, and the great immunogenicity of TSA and TAA in EAC-bearing animals show the existence of potent specific rejection antigens (TSRA and TARA) among the obtained TSA and TAA. The amount of TSA and TAA isolated from the EAC cell plasma membrane is 6 ng/10(6) cells, representing 1 part per 43,000 of the cell proteins.
Subject(s)
Antigens, Neoplasm/isolation & purification , Carcinoma, Ehrlich Tumor/immunology , Animals , Cell Membrane/immunology , Chromatography, Affinity , Immunologic Techniques , MiceABSTRACT
It is possible to obtain antisera to cancer-specific antigens of mouse Ehrlich ascites cancer (EAC) cells when chimera rabbits previously made unresponsive to immunological stimulation by normal mouse cells antigens are immunized with mouse cancer cells. Employing this specifically anti-cancerous immunoserum this work shows that EAC cells and TC-SV40 cells contain cross-reacting cancer-specific antigens. Exogenous DNA from EAC cells is demonstrated as being able to display its coded information when it is incorporated to endogenous TC-SV40 cell DNA whose activity has been previously inhibited by 5'-bromodeoxyuridine.
Subject(s)
Antigens, Neoplasm/analysis , DNA, Neoplasm/metabolism , Transcription, Genetic , Animals , Bromodeoxyuridine/pharmacology , Carcinoma, Ehrlich Tumor/immunology , Cross Reactions , Immunoenzyme Techniques , In Vitro Techniques , Mice , Rabbits , Simian virus 40/immunologyABSTRACT
An equipment simulating the pharmacodynamics of anti-cancer drugs under time lapse microcinematography is described. It consists of an inverted microscope furnished with an incubating chamber, a motion-picture camera, an intervalometer, a gradient forming device and peristaltic pump. In this work, the equipment is used to study the effect of methotrexate (MTX) on cultures of HeLa cells and HeLa cell-sensitized lymphoblasts. MTX inhibits cell reproduction of both, HeLa cells and lymphoblasts, prolongs twice the generation time of the cancer cells, and produces a progressive cell degeneration. MTX produces also an early degeneration of lymphoblasts. All these data are studied by individual frame analysis of the films.
Subject(s)
Lymphocytes/drug effects , Methotrexate/pharmacology , Cell Division/drug effects , Cell Survival/drug effects , Cells, Cultured , HeLa Cells/drug effects , HumansABSTRACT
The immunogenicity and anti-tumor activity of tumor-specific and tumor-associated antigens (TSA and TAA) isolated from the Ehrlich ascites cancer (EAC) cell plasma membrane is assayed, and compared with the action of several crude antigenic preparations. Control animals die 26 days after the EAC cells inoculation. Crude antigenic preparations produce a delay of EAC development, but all the animals finally die. The purified TSA and TAA are active at very small amounts, and inhibit completely and permanently the tumor development. The procedure shows a new approach to cancer specific active immunotherapy.
Subject(s)
Antigens, Neoplasm/immunology , Carcinoma, Ehrlich Tumor/immunology , Animals , Antibody Formation , Antigens, Neoplasm/administration & dosage , Carcinoma, Ehrlich Tumor/therapy , Cell Membrane/immunology , Female , MiceABSTRACT
Employing the pharmacologic simulator under time lapse microcinematography described elsewhere, the authors study in this work the rescue treatment with high doses of methotrexate (MTX) followed by leucovorin rescue 6 and 24 hours after MTX addition. The experiments were done on mixed cultures of HeLa cells and HeLa cell-sensitized human lymphoblasts. MTX alone produces an extensive cell degeneration, that is never complete, 2300 micrograms/ml leucovorin have no toxic effect neither upon HeLa cells nor lymphoblasts. Higher amounts of leucovorin stimulate the growth of the neoplastic cells, but the lymphoblasts are not affected. Leucovorin added 6 or 24 hours after MTX inhibits the mitotic activity completely for 54 to 62 hours. Neoplastic cells death rate is never higher than 20 per 100. Lymphoblasts die much earlier reaching 50 per 100 after 50-58 hours. Cell degeneration is accompanied by large morphological and morphodynamic modifications. Rescue treatment in vitro, under condition simulating in vivo kinetics of the drugs is not able to eliminate the cancer cells. Actually, leucovorin stimulates the growth of the neoplastic cells and rescues great part of them out from the G0 state into the active cell cycle when leucovorin is added 6 or 24 hours after MTX administration, no growth stimulus is observed and the rescue effect is not produced; in the case of the lymphoblasts this is due to the fact that highest toxic of MTX on them takes place during the second elimination phase of the drug.
Subject(s)
Leucovorin/pharmacology , Lymphocytes/drug effects , Methotrexate/pharmacology , Cells, Cultured , Drug Interactions , HeLa Cells/drug effects , Humans , Mitosis/drug effectsABSTRACT
The effect of methotrexate (amethopterin) upon the MMT cell line was studied by time-lapse microcinematography, the plasma levels obtained after systemic administration of maximum tolerated doses of the drug in man being simulated in vitro. Cells in the logarithmic growth phase (large growth fraction population) were widely affected, although enough drug-resistant cells remained to regenerate the cell colony. Cells in the preconfluent growth phase (small growth fraction population) were less effected, because many cells were arrested at the GO-hase, outside the cell cycle. A drug-resistant colony always developed, making the drug therapy useless. The experiments showed that rescue treatment with leucovorin (citrovorum factor of folinic acid) was not effective either, because, at least on our experimental conditions, recovery of the mitotic activity was more rapid and the number of degenerating cells smaller with rescur treatment than with the conventional treatment. The results also suggested a new mechanism of methotrexate action in addition to the classic one of folic acid inhibition, which might consist in the inhibition of the production of formyl-methionyl-tRNA, part of the initiation complex in protein biosynthesis.
Subject(s)
Mammary Neoplasms, Experimental/drug therapy , Methotrexate/pharmacology , Animals , Cell Cycle/drug effects , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , HeLa Cells/drug effects , Humans , Leucovorin/pharmacology , Methotrexate/toxicity , Mice , Mitosis/drug effects , Motion PicturesABSTRACT
The amount of rough endoplasmic reticulum (RER) and associated polyribosomes changes during the cell cycle. We describe here the amount and activity of free and RER-bound ribosomes of Ehrlich ascites cancer cells. RER does not exist during the G1, G2 and M phases, it appears in the early S phase and decreases in the second half of S. The formation of RER during the S phase and the S phase RER-found ribosomes may be related to the biosynthesis of histones, some fundamental non-histonic nuclear proteins, and enzymes involved in DNA replication and several metabolic reactions.
Subject(s)
Carcinoma, Ehrlich Tumor/ultrastructure , Endoplasmic Reticulum/ultrastructure , Ribosomes/ultrastructure , Animals , Autoradiography , Cell Cycle , Interphase , Mice , Microscopy, Electron , Polyribosomes/ultrastructureABSTRACT
The antiblastogram allows the specific determination within a few days of the effect of single and combined chemotherapeutic agents upon the cells of a given tumor, informing about their action, summation, antagonism and synergism. It permits to evaluate the activity of both hydrosoluble and liposoluble products, giving information about the possible activation or inactivation of chemotherapeutic agents by different substances and subcellular fractions.