retinol-acetate and Cell-Transformation--Neoplastic

It has been suggested that electromagnetic (EM) fields can act as co-promoters during neoplastic transformation. To examine this possibility, we studied the effects of 0.8-, 8-, 80-, and 300-microT 60-Hz electromagnetic (EM) fields in INITC3H/10T1/2 mouse fibroblast cells. These cells are transformed carcinogenically by methylcholanthrene, but the neoplastic phenotype can be suppressed indefinitely by the presence of retinyl acetate (RAC) in the culture medium. The effects of EM field exposures were examined at three stages: (1) before initiation of transformation (i.e., RAC in the culture media); (2) early in the transformation process (4 days after withdrawal of RAC); and (3) at full of neoplastic transformation (10 days after withdrawal of RAC). EM field exposures induced significant increases in protein levels for hsp70 and c-Fos and in AP-1 binding activity. EM fields induced phosphorylation of MAPK/ERK1/2 before the onset of transformation, but these increases diminished during the transformation process. No phosphorylation in the other major extracellular stress pathway, SAPK/JNK, was detected in cells exposed to EM fields at any time before, during, or after neoplastic transformation. Human cells HL60, MCF7, and HTB124, exposed to EM fields, also showed MAPK/ERK1/2 phosphorylation. Cells treated with the phorbol ester, TPA, served as positive controls for AP-1 activation, c-Fos protein synthesis, and ERK1/2 phosphorylation. There was no indication that EM fields affected the rate of cell transformation or acted as a co-promoter, under the conditions of this study.

Topics: Animals; Blotting, Western; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; DNA; Electromagnetic Fields; Electrophoresis, Agar Gel; Fibroblasts; HSP110 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-fos; Retinyl Esters; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Vitamin A

Scanning electron microscopy and freeze fracture electron microscopy were used to characterize membrane ultrastructural differences between parental, C3H/10T1/2, and carcinogen-initiated, INIT C3H/10T1/2, cells and treatments with retinyl acetate. The intramembranous particle distribution on the E-face was detected and quantitated by the methods of automated image analysis to obtain statistically meaningful numerical characteristics of intramembranous particle size and density. Subtle differences were found when no differences were apparent by light microscopy or by scanning electron microscopy. Initial retinyl acetate treatment caused a significant increase of the intramembranous particle size in parental cells. Intramembranous particle density increased for retinyl acetate treatment in parental and INIT cells and in INIT cells previously maintained but withheld from retinyl acetate. Intramembranous particle distribution analysis includes the interparticle distance of nearest neighbors and the randomness of the distribution by the differential density distribution function, which compares the observed sample to Poisson modified for particle size. These measures show that the three cell groups that have been treated with retinyl acetate have a more even distribution of intramembranous particles than was found for untreated parental cells. The relationship between the freeze fracture morphology and the biological responses to retinyl acetate treatment is discussed.

Topics: Animals; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Diterpenes; Freeze Fracturing; Membrane Proteins; Mice; Mice, Inbred C3H; Microscopy, Electron, Scanning; Retinyl Esters; Vitamin A

The effect of three retinoids, 13-cis-retinoic acid, arotinoid ethyl sulfone and retinal acetate, on methylcholanthrene (MCA) induced transformation of 10T1/2 cells was studied. It appears that 13-cis-retinoic acid and arotinoid ethyl sulfone prevent transformation in a direct way at the last stage of carcinogenesis. Retinal acetate, however, requires cell-to-cell contacts to inhibit the transformation of 10T1/2 cells.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; Methylcholanthrene; Mice; Retinoids; Retinyl Esters; Tretinoin; Vitamin A

The effect of retinyl acetate (RAC) on the activity of cAMP-dependent protein kinase (PKA) was studied in mouse 10T1/2 cells. The studies revealed that normal 10T1/2 cells had about 13-fold more PKA activity than did methylcholanthrene-transformed cells (MCA cells). The addition of RAC to MCA cells increased the activity of PKA about 3-fold as measured by the in vitro phosphorylation of a specific site in H1 histone (site A) or Kemptide. The increased PKA activity coincided with a reduction in the rate of cell replication of MCA cells, about 24 hr after exposure to the retinoid. Addition of forskolin to RAC-treated MCA cells resulted in a further reduction in the rate of cell replication, and this suggested that the enhanced PKA activity was also capable of action in vivo. To test this notion, MCA cells were grown with and without RAC, and the phosphorylation of the H1 histone at site A, a site known to be phosphorylated by PKA in cells treated with hormones or other agonists which activate PKA, was studied in vivo. RAC, by itself, was capable of causing an increase in the phosphorylation of the H1 histone at site A, demonstrating that the retinoid-mediated increase in PKA activity was sufficient to cause the enhanced phosphorylation of a known substrate.

Topics: Animals; Cell Nucleus; Cell Transformation, Neoplastic; Cells, Cultured; Cytosol; Diterpenes; Histones; Mice; Peptide Mapping; Phosphopeptides; Protein Kinases; Retinyl Esters; Vitamin A

Retinyl acetate (RAC) prevents neoplastic transformation of carcinogen-exposed C3H/10T1/2 C18 cells, and has allowed the isolation of cells having the properties expected of initiated cells. After removal of RAC from logarithmically growing initiated cells, cultures first become confluent and growth arrested, as occurs with their normal counterparts. This is followed by an increase in thymidine labelling index and finally by morphological transformation on days 15 and 23, respectively, after seeding and drug removal. The increase in labelling index is the earliest indication yet seen of transformation taking place in these cultures. Phosphorylation of three nonionic detergent-soluble proteins was observed to be correlated with the increase in labelling index. Using two-dimensional gel electrophoresis, a protein of 35 kd (pp35(1)) and one of 38 kd (pp38) were seen to become more heavily phosphorylated in association with the increase in labelling index. A second protein of 35 kd (pp35(2)) was only detected in isolated transformed cells and is phosphorylated in an alkali-resistant manner consistent with phosphorylation of tyrosine residues. Alkali-resistant phosphorylation of pp35(2)) could be eliminated in transformed cells by treatment with RAC. Phosphorylation of pp35(1)) appears to be a permanently acquired characteristic of these cells, and cannot be made to revert. Phosphorylation of pp38, as indicated by isoelectric point, is reduced by RAC treatment. These studies implicate changes in protein phosphorylation in loss of growth control, and suggest that the cancer chemopreventive action of retinoids may also be mediated at this level.

Topics: Animals; Calcium; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Diterpenes; ErbB Receptors; Mice; Molecular Weight; Phosphates; Phosphoproteins; Phosphorylation; Proteins; Receptors, Cell Surface; Retinyl Esters; Tetradecanoylphorbol Acetate; Vitamin A

We have demonstrated that non-toxic concentrations of retinoids can cause a dose-dependent inhibition of 3-methylcholanthrene-induced transformation of C3H/10T1/2 cells. On removal of the retinoid, transformed foci appear after a latent period of about four weeks at the same frequency as observed in controls treated with carcinogen only. Reasoning that this activity is compatible with the stabilization of the carcinogen-initiated state, we have succeeded in isolating from carcinogen-treated cultures a cell line which in the presence of retinyl acetate is similar to the parental 10T1/2 cells, but without retinyl acetate transforms at a high frequency after a latent period of about four weeks. Retinyl acetate treatment of this cell line (INIT/10T1/2) and the parental 10T1/2 cells induces an ultra-normal phenotype. In retinoid-deprived INIT/10T1/2 cells, the first sign of transformation (an increased thymidine-labelling index) occurs 16 days after retinyl acetate removal. We have detected by two-dimensional electrophoresis that concomitant with this there is an increase in phosphorylation of a protein of Mr 34 000 (34K) which may be associated with the cytoskeleton. This phosphoprotein has been found in all transformed lines examined. A second phosphoprotein, of about 38K, has also been detected in transformed cells. Retinyl acetate treatment of transformed cells alters the isoelectric point of this protein, a change compatible with decreased phosphorylation. Alkali-resistant phosphorylation, presumably on tyrosine, has been found on a second 34K protein of transformed cells. Retinyl acetate treatment specifically decreases this phosphorylation. Mechanisms for the altered tyrosine phosphorylation induced by retinyl acetate are as yet unresolved, but the decrease could be due to altered levels of substrate, kinase or phosphorylase. In view of the apparent role of tyrosine kinases as mediators of growth factors and as oncogene products, we consider the activity of retinoids as modulators of tyrosine phosphorylation to be of great potential significance.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Diterpenes; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; In Vitro Techniques; Mice; Mice, Inbred C3H; Neoplasm Proteins; Phosphorylation; Retinoids; Retinyl Esters; Time Factors; Vitamin A

Invasion of chick chorioallantoic membrane (CAM) organ cultures by rat 3Y1 cells transformed by the highly oncogenic human adenovirus type 12 (3Y1/12-10 cells) was inhibited by several retinoids tested. The anti-invasive activity of the retinoids was dependent on retinoid concentration and continuous (4 d) exposure of the CAM. The 50% retinoid dose (dose effective in achieving a response in half of the organ cultures) that inhibited invasion was 0.85 micrograms/ml of retinol palmitate, 0.39 micrograms/ml of retinoic acid, or 0.16 micrograms/ml of retinol acetate. This dose was of the same order of magnitude as that which induced CAM differentiation, and was three- to fourfold less than the dose that caused cytotoxic damage of CAM. In addition, the retinoids inhibited 3Y1/12-10 cell growth by approximately 40% at levels over 10-fold higher than those needed for anti-invasion activity. The findings suggest that the anti-invasive activity of retinoids was at least partly due to direct induction of cell differentiation of the CAM host tissue.

Topics: Adenoviruses, Human; Allantois; Animals; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Chick Embryo; Chorion; Diterpenes; Extraembryonic Membranes; Neoplasm Invasiveness; Organ Culture Techniques; Rats; Retinoids; Retinyl Esters; Tretinoin; Vitamin A

Topics: Animals; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Diterpenes; Fibroblasts; Mice; Mice, Inbred C3H; Neoplasm Seeding; Retinoids; Retinyl Esters; Vitamin A

Retinyl acetate has been previously shown to inhibit carcinogen-induced neoplastic transformation in 10T1/2 cells and to accentuate many aspects of the nontransformed phenotype. Scanning electron microscopy of logarithmic phase 10T1/2 cells treated for 3 days with 0.3 micrograms/ml retinyl acetate revealed that this treatment caused extensive flattening of cells to the plastic substrate. In contrast the tumor promoter tetradecanoyl phorbol acetate, which antagonizes the antineoplastic activity of retinyl acetate, caused cell rounding and completely inhibited the action of retinyl acetate on cell morphology. During this same time course, the formation of microfilament bundles was also found to be modulated by retinyl acetate. Transmission electron micrographs of unsectioned peripheral regions of flattened cells showed that while the unit density of microfilament bundles was not influenced, the thickness of bundles, particularly those with a diameter of 100 nm or more, was increased by retinyl acetate. Tetradecanoyl phorbol acetate had little effect on microfilament bundle diameters but did partially antagonize the action of retinyl acetate. To determine if this increase was associated with an increase in total actin/cell, total cell proteins, and proteins not extractable by glycerol-triton extraction, were subjected to sodium dodecylsulfate/ polyacrylamide gel electro-phoresis. It was found that while total cellular actin was not increased by retinyl acetate, the proportion of nonextractable actin (which includes microfilament bundles) increased from 65% to 88% of total actin. This increase was not inhibited by inhibitors of protein or RNA synthesis. These studies again demonstrate that retinyl acetate accentuates the nontransformed phenotype of 10T1/2 cells; it is hypothesized that these actions are related to the antineoplastic activity of retinoids.

Topics: Actins; Animals; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Cytoskeleton; Diterpenes; Mice; Microscopy, Electron; Retinyl Esters; Tetradecanoylphorbol Acetate; Vitamin A

The effects of retinyl acetate and all-trans-retinoic acid on the growth rate, saturation density, and cytoplasmic underlapping of normal and carcinogen-initiated C3H/1OT1/2 cells were examined. Retinyl acetate (a) decreased the saturation density by as much as 45%, (b) had no effect on the growth rate, (c) reduced cytoplasmic underlapping of adjacent cells by 55 to 85%, and (d) inhibited neoplastic transformation by methylcholanthrene. The effects were dose dependent and not significantly affected by the serum concentrations over the range of 2.5 to 10%. In contrast, all-trans-retinoic acid (a) decreased the saturation density as effectively as retinyl acetate, but only in medium containing 10% serum; (b) significantly reduced the growth rate of cells at low density, especially at low serum concentrations; (c) had no effect upon cytoplasmic underlapping; and (d) enhanced transformation at nontoxic concentrations in medium containing 5% serum but had no effect in 10% serum. We conclude that the effects of retinoids on the growth rate and saturation density of cells in culture may not be relevant to their inhibition of neoplastic transformation. Rather, we interpret the results of our experiments on cytoplasmic underlapping as an indication that retinoids inhibit transformation by stabilizing and/or enhancing cell surface receptors involved in cell-cell contact-dependent formation of a stable monolayer.

Topics: Animals; Cell Cycle; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; Embryo, Mammalian; Kinetics; Methylcholanthrene; Mice; Mice, Inbred C3H; Retinyl Esters; Tretinoin; Vitamin A

Vitamin A and its analogues (retinoids) affect normal and malignant hematopoietic cells. We examined the effect of retinoids on the clonal growth in vitro of myeloid leukemia cells. Retinoic acid inhibited the clonal growth of the KG-1, acute myeloblastic leukemia, and the HL-60, acute promyelocytic leukemia, human cell lines. The KG-1 cells were extremely sensitive to retinoic acid, with 50% of the colonies inhibited by 2.4-nM concentrations of the drug. A 50% growth inhibition of HL-60 was achieved by 25 nM retinoic acid. Complete inhibition of growth of both leukemia cell lines was seen with 1 microM retinoic acid. Exposure of KG-1 cells to retinoic acid for only 3-5 d was sufficient to inhibit all clonal growth. The all-trans and 13-cis forms of retinoic acid were equally effective in inhibiting proliferation. Retinal, retinyl acetate, and retinol (vitamin A) were less potent inhibitors. Clonal growth of the human K562 and mouse M-1 myeloid leukemic cell lines was not affected by 10 microM retinoic acid. Retinoic acid also inhibited the clonal growth of leukemia cells from five of seven patients with acute myeloid leukemia. Retinoic acid at concentrations of 5 nM to 0.3 microM inhibited 50% clonal growth, and 1 microM retinoic acid inhibited 64-98% of the leukemic colonies. The inhibition of clonal growth of KG-1 and HL-60 cell lines and of leukemic cells from two patients was not associated with the presence of a specific cytoplasmic retinoic acid-binding protein. Our study suggests that retinoic acid may prove to be effective in the treatment of human myeloid leukemia.

Topics: Animals; Carrier Proteins; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Cytosol; Diterpenes; Dose-Response Relationship, Drug; Humans; Leukemia, Myeloid, Acute; Mice; Receptors, Retinoic Acid; Retinyl Esters; Tretinoin; Vitamin A

A clone of 3-methylcholanthrene-treated 10T1/2 cells has been isolated which possesses basic characteristics expected of "initiated" cells. In the presence of retinyl acetate, this clone exhibits contact inhibited growth control and is morphologically indistinguishable from the parental 10T1/2 cell line. Removal of retinyl acetate in vitro results in neoplastic transformation after a latent period of 3 weeks. The classical 10T1/2 transformation system was reconstructed by coculturing normal and "initiated" 10T1/2 cells formed either Type II or Type III foci after a latent period of 3-4 weeks, and an additional 22% formed Type I foci. Treatment of "initiated" 10T/2 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate accelerated the formation of transformed foci in the coculture system by reducing the length of the latent period to less than 3 weeks. Injection of 10(6) "initiated" cells/mouse s.c. into nude mice resulted in the appearance of progressively growing fibrosarcomas after a latent period of 5-7 weeks. Dietary supplementation with 4-hydroxyphenyl-retinamide prevented tumor formation; after drug withdrawal, tumors developed in all surviving mice after 6 weeks. We believe this cell line possesses all characteristics expected of "initiated" cells. With this new cell line, designated INIT/10T1/2, we can now study the early biochemical changes in growth control mechanisms resulting in neoplastic transformation and the mechanism(s) of chemoprevention of cancer by vitamin A.

Topics: Animals; Cell Line; Cell Separation; Cell Transformation, Neoplastic; Cocarcinogenesis; Contact Inhibition; Diterpenes; Methylcholanthrene; Mice; Mice, Inbred C3H; Mice, Nude; Neoplasm Transplantation; Retinyl Esters; Tetradecanoylphorbol Acetate; Vitamin A

Topics: Animals; Cell Adhesion; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Diterpenes; Methylcholanthrene; Mice; Mice, Inbred C3H; Retinaldehyde; Retinyl Esters; Tretinoin; Vitamin A

Pancreatic carcinomas have been induced in Wistar and W/LEW rats by administration of total azaserine doses of 150-520 mg/kg by injection or oral routes over periods of 5-52 weeks. The latent period for development of invasive carcinomas was 1-2 years, but focal abnormalities in acinar cells appear earlier. The incidence of carcinomas varied with total dose, route, and schedule of azaserine administration. The spectrum of histologic patterns of the carcinomas included well and poorly differentiated acinar cell, ductlike, and undifferentiated carcinomas. Rats fed a purified diet developed more pancreatic neoplasms than rats fed a commercial laboratory chow. Selective feeding of these diets during the administration of carcinogen and following completion of carcinogen treatment indicated that the inhibitory effect of chow on pancreatic carcinogenesis was exerted during the postinitiation phas. Supplementation of diet with 0.025% retinyl acetate during the postinitiation phase also inhibited the progression of azaserine-induced lesions in the pancreas.

Topics: Adenocarcinoma; Animals; Azaserine; Carcinoma; Cell Transformation, Neoplastic; Cytoplasmic Granules; Diet; Diterpenes; Enzyme Precursors; Male; Neoplasms, Experimental; Pancreatic Neoplasms; Rats; Rats, Inbred Lew; Rats, Inbred Strains; Retinyl Esters; Vitamin A

Various natural and synthetic retinoids have been studied for their activity in two biological systems: (a) their activity as inhibitors of methylcholanthrene-induced neoplastic transformation in the C3H/10T1/2 clone 8 mouse fibroblast line (System 1); and (b) their ability to increase the degree of adhesion of C3H/10T1/2 clone 8 cells to a plastic substrate (System 2). These activities were then compared with their known activity in maintaining epithelial differentiation (System 3). With the notable exception of retinoic acid and 13-cis-retinoic acid, which were inactive in Systems 1 and 2, an excellent correlation was observed between activities in Systems 1 and 3 for retinyl acetate, N-(4-hydroxyphenyl)retinamide, retinylidene dimedone, N-ethylretinamide, and N-benzoylretinylamine. Compounds shown to be inactive in System 1 had little or no activity in System 2. However, the ability of retinoids to cause increased adhesion could not be correlated with Systems 1 or 3 in all cases. For instance, retinyl acetate was highly active in Systems 1, 2, and 3, whereas retinylidene dimedone was highly active in Systems 1 and 3 but weakly active in System 2. Conversely, N-(4-hydroxyphenyl)retinylamide was highly active in Systems 1 and 3 but caused a decrease in System 2. The lack of activity of 3 but caused a decrease in System 2. The lack of activity of retinoic acid isomers in the C3H/10T1/2 clone 8 system is paradoxical and may provide important information on requirements for their activation and/or transport.

Topics: Amides; Animals; Cell Adhesion; Cell Line; Cell Transformation, Neoplastic; Diterpenes; Fenretinide; Isotretinoin; Methylcholanthrene; Mice; Neoplasms, Experimental; Retinoids; Retinyl Esters; Structure-Activity Relationship; Tretinoin; Vitamin A

Topics: Animals; Antineoplastic Agents; Cell Division; Cell Line; Cell Transformation, Neoplastic; Diterpenes; DNA, Neoplasm; Mammary Neoplasms, Experimental; Melanoma; Neoplasm Proteins; Neoplasms, Experimental; Retinol-Binding Proteins; Retinyl Esters; Sarcoma, Experimental; Tretinoin; Vitamin A