George E. Milo

Fatty acid specificity in the inhibition of cell proliferation and its relationship to lipid peroxidation and prostaglandin biosynthesis

Primary cultures of smooth muscle cells were established from the medial layer of guinea pig aorta. Cells at passage level 4 were treated with different series of fatty acids belonging to the n-9, n-6 and n-3 families. Lipid peroxidation was measured by the thiobarbituric acid assay and prostaglandin biosynthesis was measured by the radioimmunoassay of PGE and 6-keto-PGF1α. Cell proliferation was estimated from the total cell number of cultures seeded at low density. 18∶1(n-9) did not form lipid peroxides and this fatty acid stimulated cell proliferation. All fatty acids which generated lipid peroxides inhibited cell proliferation, but inhibition was correlated with the degree of lipid peroxidation only in the n-9 fatty acid family. 22∶4(n-6) and 22∶6(n-3) inhibited prostaglandin biosynthesis. 18∶2(n-6), 18∶2(n-9), 18∶3(n-3), 20∶2(n-9), 20∶3(n-3) and 20∶5(n-3) had no effect on prostaglandin biosynthesis. 18∶3(n-6), 20∶3(n-6) and 20∶4(n-6) generated prostaglandins. 20∶3(n-9) generated metabolites with prostaglandin immunoreactivity. The inhibition of cell proliferation did not correlate with enhanced or inhibited prostaglandin synthesis. The inhibition of cell proliferation was related to the structures of the different polyunsaturated fatty acid families decreasing in the order n-9>n-6>n-3. Eicosatrienoic acids were the most effective inhibitors of cell proliferation in each fatty acid family and 20∶3(n-9) was the most potent eicosatrienoic acid. These data show that specific as yet unrecognized products of fatty acid metabolism are responsible for the inhibition of cell proliferation.

Polyunsaturated fatty acids, vitamine E, and the proliferation of aortic smooth muscle cells

Smooth muscle cell cultures were obtained from the aortas of prepubertal guinea pigs. Cell proliferation in these cultures was inhibited by 8,11,14-eicosatrienoic acid, 5,8,11,14-eicosatetraenoic acid, and their prostaglandin E derivatives, PGE1 and PGE2. Prostaglandin F derivatives, PGF1α and PGF2α, stimulated cell proliferation. Cell proliferation was also inhibited by 5,8,11-eicosatrienoic acid and 11,14,17-eicosatrienoic acid. The monoene and diene precursors of the triene acids, 9-octadecenoic acid and 9,12-octadecadienoic acid, did not inhibit cell, proliferation. Indomethacin alone had no effect on cell proliferation, and indomethacin did not suppress the inhibition of cell proliferation with a triene acid. The antioxidant α-naphthol alone stimulated cell proliferation and suppressed prostaglandin E formation. α-Naphthol in the presence of either triene or tetraene acids also stimulated cell proliferation and suppressed prostaglandin E formation. The antioxidants butylated hydroxy toluene and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid either alone or in the presence of triene and tetraene acids stimulated cell proliferation and had no effect on prostaglandin E formation. Vitamin E either alone or in the presence of triene or tetraene acids stimulated cell proliferation and had no effect on prostaglandin E formation. More prostaglandin E was formed from 8,11,14-eicosatrienoic acid than from 5,8,11,14-eicosatetraenoic acid in the presence of antioxidants. Vitamin E suppressed the inhibitory effects of both PGE2 and palmitic acid on cell proliferation. The cyclic nucleotide phosphodiesterase inhibitors, caffeine and papaverine, suppressed the stimulatory effect of vitamin E on cell proliferation and enhanced the inhibitory effect of a triene acid on cell proliferation. Substrate and inhibitor specificities are consistent with the oxidative regulation of cell proliferation through the formation of hydroperoxy fatty acids. We propose that hydroperoxy fatty acids may regulate both cyclase and cyclic nucleotide phosphodiesterase enzymes through sulfhydryl-disulfide interconversions. We suggest that this regulatory mechanism may help to explain the acculation of 5,8,11-eicosatrienoic acid in essential fatty acid deficiency, the effects of antioxidants on cell proliferation, and one of the several effects of polyunsaturated fatty acids in proliferative disorders such as cancer and atherosclerosis.

The toxicological evaluation of the mycotoxins T-2 and T-2 tetraol using normal human fibroblasts in vitro

Abstract Normal human fibroblasts in vitro were used as a system to examine the cellular effects of potentially toxic compounds. In addition to tests for viability, indices of toxicity were included which would demonstrate functional and structural alterations associated with specific subcellular components. Fibroblast cultures derived from human neonatal foreskin were treated with various doses of the mycotoxins T-2 and T-2 tetraol. Methods for demonstrating viability showed T-2 to be 10-fold more toxic than its hydroxylated derivative, T-2 tetraol. Both compounds induced a dose-dependent reduction in protein and scheduled DNA synthesis, as well as an induction of unscheduled DNA synthesis (DNA repair). However, no qualitative alterations in the activity of cytoplasmic, lysosomal or membrane-associated enzymes were observed with T-2 or T-2 tetraol-treated cells. Transmission electron microscopy revealed structural alterations predominantly associated with the nucleus and the endoplasmic reticulum with associated ribosomes. Multiple biochemical parameters with ultrastructural and cytotoxicity studies were therefore effective in demonstrating the mechanisms as well as the degree of toxicity induced by these chemical compounds.

Enhanced expression of the 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase gene in human breast tumor cells

The expression of the 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase) gene in human breast tumors was evaluated at the level of the single cell to better understand how breast tumor cells regulate expression in response to oxidative stress. Compared to normal breast ductal cells, the level of 8-oxo-dGTPase expression in the breast tumor cells increased from non-detectable levels in normal breast to expression in 30-85% of the tumor cells in individual tumors. There was no significant association between 8-oxo-dGTPase expression and tumor grade and metastatic malignancy. The upregulation of 8-oxo-dGTPase was not directly linked to the expression of cyclins D1 and D3, estrogen receptor, p53, Ki-67 and c-erbB-2, which are genes involved in cell cycle regulation and tumor growth. The elevated expression of 8-oxo-dGTPase in human breast ductal carcinoma cells appears to be a general characteristic of breast tumors and may provide the tumor cell with a cellular defense mechanism to prevent the incorporation of 8-hydroxy-deoxyguanosine during DNA replication.

Characterization of human cells transformed by chemical and physical carcinogens in vitro

SummarySeveral different classes of chemical carcinogens induced the transformation of human fibroblasts grown in vitro. Characteristics of the events that occur from time of treatment through the expression of neoplastic transformation are presented. The S-phase appeared to be the portion of the cell cycle most vulnerable to insult. Staging of the cells by blocking them in G1 before releasing them to proceed through scheduled DNA synthesis (S) was required to induce reproducible transformation. Compounds such as insulin were added to the cells upon release from the block to sensitize the cells to the carcinogen that was added during S. Growth of the transformed cells as distinct from nontransformed cells was promoted by growth in medium supplemented with 8X nonessential amino acids. Carcinogen-treated cells in the early stage of transformation exhibited abnormal colony morphology and were able to grow at 41°C, in air atmosphere, and in medium supplemented with only 1% serum. In addition, the transformed cells were insensitive to KB cell lysate and exhibited density independent, as well as anchorage independent, growth (i.e., growth in 0.33% agar). Cells that grew in soft agar also produced undifferentiated mesenchymal tumors in preirradiated nude mice.

Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro

SummaryFetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum.Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-β-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 μg per ml of progesterone. The addition to non-supportive sera of 0.1 μg per ml of 17-β-estradiol or hydrocortisone made these sera supportive of cell growth.Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro.

Fatty acids and the selective alteration of in vitro proliferation in human fibroblast and guinea-pig smooth-muscle cells

SummaryHuman-foreskin fibroblast (HF) and guinea-pig aorta smooth-muscle (SM) cultures were treated with several saturated and unsaturated fatty acids. Relative plating efficiencies were used to determine the proliferative response to each treatment. At low concentrations (16 to 18 μm), proliferation in HF cultures was inhibited by 8,11,14-eicosatrienoic acid (20:3), and stimulated by both 5,8,11,14-eicosatetraenoic acid (20:4) and 9-octadecenoic acid (18:1). At these levels, proliferation in SM cultures was unchanged by 20:3, inhibited by 20:4, and enhanced by 18:1. At higher concentrations (80 to 90 μm), HF cultures were inhibited by all three unsaturated fatty acids. At these same concentrations, proliferation in SM cultures was inhibited by 20:3 and 20:4, whereas 18:1 continued to stimulate proliferation. Thus proliferative response was a specific effect of the fatty acid used, its concentration, and the cell line involved. Further treatment of SM cultures by tetradecanoic acid (14:0), hexadecanoic acid (16:0), and octadecanoic acid (18:0) showed that their relative abilities to inhibit cell proliferation increased with increasing chain length. Concentrations required for the effective inhibition of proliferation in SM cultures by 14:0, 16:0 and 18:0 were 220 μm, 95μm and 18μm, respectively. The fatty acids used in these studies are all endogenous components of sera used as growth supplements in in vitro systems. Their roles as prostaglandin and hydroperoxy fatty-acid precursors (20:3 and 20:4), inhibitors of prostaglandin biosynthesis (18:1), or as calcium ionophores (14:0, 16:0, and 18:0) may allow them to function as endogenous controls of cell proliferation.

Conditions for transformation of human fibroblast cells: an overview

Low passage (low population doubling) human diploid fibroblasts respond to carcinogen and mutagen treatment, with higher passage level human cells remaining refractory to the insult. A cell cycle dependency for an optimize response to the carcinogen of competent responsive low passage cells is associated with early S phase. The process of fixation of the damage in dividing young cells could be more efficient due to intrinsic sensitivity of young cells towards carcinogens. However, specific DNA-carcinogen adduct analysis does not reveal any qualitative or quantitative difference. These low passage carcinogen initiated human cells progress towards the expression of a malignant phenotype. There is little evidence to suggest that these abnormal phenotypes exhibit an infinite lifespan using the selection pressures for isolation of the transformed phenotypes. However, the lifespan of these treated cells is extended beyond those of the untreated cells. In conclusion, criteria can be established to measure the expression of progression of these carcinogen initiated cells towards a malignant phenotype.

Inhibition of carcinogen-induced cellular transformation of human fibroblasts by drugs that interact with the poly(ADP-ribose) polymerase system: Initial evidence for the development of transformation resistance

Two types of interactions of 13 drugs with human fibroblasts were determined: (a) I 50 of nuclear poly(ADP‐ribose) polymerase, as assayed with isolated nuclei in vitro, and (b) the non‐toxic concentration of drugs that prevented carcinogen‐induced cell transformation of intact fibroblasts (RCF1). In general, RCF1 was much lower than I 50, and one antitransformer did not inhibit the enzyme in vitro, indicating that low‐affinity enzyme inhibitory sites appear to play no role in the mechanism of prevention of cell transformation. Two enzyme inhibitors, caffeine and 1‐methylnicotinamide, exhibited no antitransforming activity. Benzamide when applied in population doubling 1 induced resistance to cell transformation in population doubling 6 by carcinogens added at this stage.

Age-related alterations in plasma membrane glycoprotein content and scheduled or unscheduled DMA synthesis

Abstract WI-38 cells of various ages and SV40-transformed WI-38 cells were examined for differences in plasma membrane composition of glycoproteins and DNA synthesis. Sialic acid per milligram of protein content of the membranes of WI-38 cells decreased with passage of time in culture. Other glycoprotein fractions and alkaline phosphatase activity disappeared in the WI-38 cells with passage of time in culture (Phase III). Studies of DNA repair correlated with changes observed in the plasma membrane glycoprotein content of WI-38 cells over a passage of time in culture were also reported. Both the extent and rate of ultraviolet-induced unscheduled DNA synthesis remained relatively constant during the passage of the WI-38 cells until late phase III. At that time the extent of unscheduled DNA synthesis was measurably reduced. The number of cells in a population of phase III cells able to perform semiconservative DNA synthesis diminished with age in culture but not to an extent capable of explaining the observed changes seen in membrane composition of semiconservative DNA synthesis during passage of the cells in culture. Cells with an extended lifespan SV40-transformed WI-38 (VA 13.2 RA) cells, did not vary in membrane composition, semiconservative DNA synthesis, or unscheduled DNA synthesis over 200 serial subpassages of the cells in culture.