Biomedical subjects
Publications and source records attributed to Y Valladares.
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.
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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.
The organism produces substances whose presence or amount indicate the existence of a neoplastic process, called biological tumor markers. They can be used in the clinical laboratory for diagnostic purposes, and also to orient about the evolution of the tumoral mass. There are about 90 tumor markers, 33 of them have studied in relation to cancer of the ovary, that are described in this work. Eight of them are the most important: CSAp, OCA, OCAA, IAP, Nagao's isoenzyme, hCG/hCG beta, hPL, and AFP.
As an example of the usefulness of biological tumor markers for the diagnosis and following up of some tumors, the author reviews briefly their application in lung cancer, breast cervix uteri, prostate cancer and acute lymphoblastic leukemia.
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.
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This work makes a brief summary of the scientific research carried out during the last fifteen years (1968-1982) in the Department of Biology and Biochemistry of Cancer (AISN, Department of Health and Consumption), identified with the Coordinated Center of Oncological Biochemistry of the CSIC (Department of Education and Science), with the valuable help of the Spanish Association Against Cancer (AECC) the first five years 1968-1972), and of the Scientific Foundation of the AECC the following ten years (1973-1982). A brief mention of the research work carried out between 1945 and 1967 is made before referring to the work done between 1968-1982. The researchers of the Department have done important studies in the fields of biochemistry, molecular biology, virology and immunology of cancer. They described for the first time the existence of immunological processes in ontogenic evolution and cell differentiation; the transfection with DNA from several kinds of tumors, including a mouse sarcoma induced by 20-methylcholanthrene (now 3-methylcholanthrene) and a human lung carcinoma; the evolution of the humoral immunological response in SWR and AKR mice along the whole life; the isolation of a polytumoral DNA virus; the carcinogenic activity of viral DNA; the establishment of a virus-transformed cell line "in vitro", the incorporation of the viral genome to the cell genome; the reverse transcription; the antiblastogram, an "in vitro" predictive assay of anti-cancer chemotherapy agents; and the isolation of tumor-specific and tumor-associated antigens.
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.
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.
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.
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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.
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.
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.
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.
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.
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.