Effect of vitamin D3 and 1,25(OH)2D3 on growth of neoplastically derived cell lines and their alkaline phosphatase activity.

Inhibitory effects of 1,25(OH)2D3 and D3 on growth of four neoplastically derived cells were observed in human acute leukemia cell culture CEM-C-1 and CEM-C-7, human cervical carcinoma cell lines C-4-1 and human epithelioid carcinoma cells of cervix HeLa S3K. Concurrently, in dexamethasone-responsive cells C-4-1 and HeLa S3K there was a 1,25(OH)2D3 and D3 induced elevation of alkaline phosphatase with 1,25(OH)2D3 showing the greater effects. It is supposed that vitamins D3-induced alkaline phosphatase activity in malignant cells, which is proposed to be a possible marker of cell differentiation, can be associated with the membrane effects of these vitamins.

Mechanism of farnesol cytotoxicity: further evidence for the role of PKC-dependent signal transduction in farnesol-induced apoptotic cell death.

Mechanism of the inhibitory effect of isoprenoid farnesol on cell proliferation has been studied in human acute leukemia CEM-C1 cells. Farnesol (20 microM) reduced the rate of radioactive label incorporation into cellular diacylglycerol (DAG) and phosphocholine, the products of degradation of phosphatidylcholine (PC), indicating inhibition of PC-specific phospholipase C after about 1 h of incubation. Inhibition of phospholipase D by farnesol at the later incubation time (about 2 h) was demonstrated by a decrease in synthesis of PC-derived phosphatidylethanol in the presence of ethanol. These effects of farnesol on PC degradation and formation of DAG were followed by apoptotic fragmentation of cellular DNA and inhibition of cell growth. Exogenous DAG reduced the level of DNA fragmentation and cell growth inhibition. Results are consistent with the involvement of cellular signal transduction in the mechanism of inhibition of cell proliferation by farnesol.

Selective farnesol toxicity and translocation of protein kinase C in neoplastic HeLa-S3K and non-neoplastic CF-3 cells.

We have reported earlier that farnesol, a 15 carbon isoprenoid, has inhibitory effects on the growth and viability of a variety of cultured cells of neoplastic derivation but is considerably less cytotoxic to cells derived from normal tissue (Cancer Lett., 79, 175-179). As part of our search for the mechanism of this observation, we have studied the effect of 20 microM farnesol on the distribution of protein kinase C (PKC) between cytosolic and membrane fractions of HeLa S3K cells and fibroblasts line CF-3. In HeLa cells farnesol caused translocation of PKC from membrane fraction to cytosol after 1h of incubation and also prevented PMA-stimulated induction of PKC translocation from cytosol to membranes. Up to 6 h of incubation, there was no effect of farnesol on PKC localization in CF-3 fibroblasts. The results point to possible involvement of PKC in the toxic effect of farnesol which occurs with some degree of selectivity depending on cell origin.

Effects of farnesol on the thermotropic behavior of dimyristoylphosphatidylcholine.

Differential scanning calorimetry (DSC) and DPH fluorescence anisotropy have been used to investigate the effects of trans-trans farnesol on the physical properties of model membranes and extracted cell lipids. Farnesol was shown to have a significant effect on the gel to liquid-crystal phase transition temperature, the enthalpy of the transition and the transition co-operativity for extruded vesicles of dimyristoylphosphatidylcholine (DMPC). The phase transition of DMPC vesicles was eliminated at 25 mol% farnesol. Farnesol decreased the fluorescence anisotropy of the lipids extracted from human leukemia line CEM-C1 cells.

Directed cell killing (apoptosis) in human lymphoblastoid cells incubated in the presence of farnesol: effect of phosphatidylcholine.

Previously reported observations have shown that trans-trans farnesol inhibits incorporation of choline into phosphatidylcholine and reduces the growth rate of the human acute leukemia CEM-C1 cell line (Melnykovych, G., Haug, J.S. and Goldner, C.M. (1992) Biochem. Biophys. Res. Commun. 186, 543-548). These findings have now been followed up in order to establish a relationship between the inhibition of phosphatidylcholine synthesis and the ensuing cell shrinkage and cell death which takes place at higher concentrations of farnesol or upon long incubation. The present results show that after incubation in the presence of farnesol the cells decrease in viability. Their nuclear DNA becomes fragmented at internucleosomal linker regions, showing characteristic pattern of bands at 180 to 200 base-pair intervals. This farnesol-induced effect was also demonstrated by flow cytometry by staining the cellular DNA with propidium iodide and was partially reversible with phosphatidylcholine.

Differences in sensitivity to farnesol toxicity between neoplastically- and non-neoplastically-derived cells in culture.

Six neoplastically-derived cell lines and three cell lines derived from normal tissues were compared for their sensitivity to isoprenoid trans-trans farnesol. Assays of cell numbers and of protein concentrations per culture revealed greater sensitivity of neoplastic cells than of the normal cells. Similar differences were obtained from the comparison of incorporation of [methyl-3H]choline into cellular lipids, with neoplastic cells showing greater inhibition than normal cells.

Farnesol inhibits phosphatidylcholine biosynthesis in cultured cells by decreasing cholinephosphotransferase activity.

The mechanism of inhibition of phosphatidylcholine (PC) biosynthesis by the isoprenoid farnesol was investigated in the human leukaemic CEM-C1 cell line. Cells were preincubated with 20 microM farnesol for up to 2 h and pulsed with [3H]choline. PC biosynthesis was inhibited to one-quarter at the step catalysed by cholinephosphotransferase (CPT). CPT activity in cellular homogenates from farnesol-treated cells was significantly decreased, but no changes in cytidylyltransferase activity or diacylglycerol concentration were observed. Measurements of CPT activity in the experiments in which farnesol was added directly to the homogenates or microsomal fractions demonstrated that farnesol did not affect CPT activity. However, cytosol from farnesol-treated samples decreased microsomal CPT activity almost twice as much as did cytosol from controls. This effect was found to be heat-stable, and disappeared after dialysis, but could not be attributed to farnesol present in the cytosol. The effect of farnesol was specific when compared with other structurally similar isoprenoids. We conclude that farnesol brings about changes in cultured cells, leading to decreased CPT activity, and thus to the inhibition of PC biosynthesis.

Growth inhibition of leukemia cell line CEM-C1 by farnesol: effects of phosphatidylcholine and diacylglycerol.

Acute leukemia cells of the established line CEM-C1 were treated during growth in serum-free medium with various concentrations of trans-trans farnesol. At concentrations ranging from 9.0 to 31.5 microM, farnesol inhibited growth of these cells without causing cell lysis. This effect was preceded by very rapid inhibition of choline incorporation in cellular lipid fraction. The growth inhibitory effect was prevented to a large extent by incubation with phosphatidylcholine or diacylglycerol.

Protein-linked isoprenoid lipids in dexamethasone-treated human lymphoid lines in culture.

Accumulation of isoprenoids was studied in two cell lines derived from acute T-cell leukemia: CEM-C7 cells, whose growth is inhibited by the glucocorticoid dexamethasone, and CEM-C1 cells, which are resistant to this steroid. Isoprenoids were measured by growing the cells in serum-free medium in the presence of lovastatin, which blocks synthesis of mevalonate, and then labeling with exogenous [3H]mevalonolactone. In both cell lines, isoprenoids associated with proteins were detected in cytoplasm, nucleus, and chromatin, and in the chromatin residue that remains after extraction of histone and nonhistone proteins. Differences in labeling were detected after treatment with dexamethasone in the CEM-C7 line, showing a decrease in the cytoplasmic fraction with a corresponding increase in both the nuclear and chromatin fractions as compared with untreated cells. No change was seen in the CEM-C1 line. In both cell lines, 25-30% of the incorporated label was released by treatment with acid or alkali. However, the majority of the label required treatment with methyl iodide for the release of organic-soluble tritiated products. After extraction with chloroform, the lipid fractions contained farnesol, geraniol, dolichols, and possibly nerolidol.

Alkyllysophospholipid influenced melanoma cell morphology is associated with decreased attachment to basement membrane.

The alkyllysophospholipid analog 1-0-octadecyl-2-0-methyl-3-phosphorylcholine (ET-18-OCH3) was examined for possible anti attachment effects on B16-F10 murine melanoma cells in vitro. At sub-lethal lipid concentrations B16-F10 cells were inhibited from attaching to reconstituted basement membrane (Matrigel) during a 45 min assay. This type of inhibition was also imparted by the isoprenoid farnesol but not by egg lysophosphatidylcholine (LPC) at concentrations up to 10 micrograms/ml. Both lipids were toxic to B16-F10 cells in the absence of bovine serum albumin (BSA), BSA (0.1%) completely protected the cells from lysis except when both lipids were combined as a mixture. Light and electron microscopy, as well as electronic sizing of cells, gave evidence of alkyllysophospholipid induced reduction in cell size which correlated well with attachment inhibition. The results suggest that alkyllysophospholipid induced reduction of cell surface area leads to inhibition of cell attachment to basement membrane which 8 with our experimental conditions, was not permanent since cells eventually attach within 24 h after treatment. The enhanced lytic effect the lysophospholipid imparts on the alkyl compound, in conjunction with the anti-attachment properties should be important areas for future research.

Effects of compound RU38486 on growth and lipid biosynthesis in glucocorticoid sensitive human leukemia line CEM-C7.

Glucocorticoid sensitive human lymphoblastoid cells CEM-C7 were examined for the effects of antiglucocorticoid RU38486 on the prevention of early dexamethasone-induced changes, including reduced cell growth, cell shrinkage and fragmentation, decrease in cell plating efficiency and incorporation of acetate into cellular lipids. When RU38486 was added no later than 24 hours after the addition of dexamethasone, it prevented the inhibition of [14C]acetate incorporation into nonsaponifiable lipids, partly reversed the decrease in plating efficiency and reduced cell fragmentation. In addition, the accumulation of dolichols in the nuclei of dexamethasone-treated cells was abolished by RU38486. These results indicate that glucocorticoid-induced changes in cellular lipids are receptor dependent and may be linked to the initiation of the apoptotic cascade.

Corticosteroid effects on lipid lateral diffusion in CEM-C1 and CEM-C7 acute lymphoblastic leukemia cells.

We have examined glucocorticoid effects on CEM-C7 and CEM-C1 subclones of a leukemic human T-cell line using fluorescence photobleaching recovery techniques. Incubation with 10(-5) M triamcinolone acetonide (TA) increased lipid lateral diffusion on steroid-sensitive CEM-C7 cells but had no effect on steroid-resistant CEM-C1 cells. CEM-C7 cells incubated in serum-free medium responded only to TA but, when fetal calf serum was added to the incubation medium, would also respond to 10(-5) M dexamethasone and hydrocortisone. Thus, glucocorticoids can cause increased lipid lateral diffusion in CEM-C7 cells, while having no effect on steroid-resistant CEM-C1 cells.

Apoptosis: mode of cell death induced in T cell leukemia lines by dexamethasone and other agents.

Glucocorticoids can mediate the destruction of thymocytes and T cell-derived leukemia cells through a mechanism known as apoptosis. The characteristic feature of apoptosis is fragmentation of DNA at internucleosomal linkers through the activity of a specific endonuclease. In this study, an attempt was made to compare dexamethasone-induced apoptosis in two T cell-derived human leukemia lines (CEM-C1 and CEM-C7) to the cell killing brought about by selected cytotoxic agents. In the CEM-C7 cell line (dexamethasone-sensitive), apoptosis was induced not only by dexamethasone but by actinomycin D, cycloheximide, and 25-OH cholesterol. In the CEM-C1 cell line (dexamethasone-resistant) cycloheximide, 25-OH cholesterol, or cell starvation could induce apoptosis. It appears that in leukemic cells apoptosis may be induced by a variety of unrelated toxic agents and is not limited to glucocorticoids.

Growth effects of regulatory peptides on human pancreatic cancer lines PANC-1 and MIA PaCa-2.

Several studies have reported effects of gastrointestinal regulatory peptides on growth of experimentally induced pancreatic neoplasms and human cancer cell lines. The growth of human pancreatic cancer lines PANC-1 and MIA PaCa-2 was characterized in vitro, and the effects of cholecystokinin, bombesin, insulin, epidermal growth factor, secretin, vasoactive intestinal peptide, and somatostatin were determined. Fetal bovine serum was required for initiation of growth in both cell lines. Growth effects of peptides were determined by incubating cells with peptides in serum-free medium after a 72-h preincubation in 10% serum-supplemented medium alone. Epidermal growth factor (3.4 x 10(-9) M) and insulin (10(-6) M) significantly (p less than 0.001) increased growth of both cell lines as determined by increases in deoxyribonucleic acid and protein. Bombesin, secretin, vasoactive intestinal peptide, and somatostatin (all 10(-8) M) did not affect growth of either cell line. Neither cholecystokinin-8 nor [Thr4, Nle7] cholecystokinin-9 altered growth in concentrations from 10(-12)-10(-6) M. Anchorage-dependent clonogenic growth of both cell lines was also not altered by cholecystokinin-8. Cholecystokinin added to cultures was degraded by separate effects of serum and cells. Addition of cholecystokinin-8 to cultures every 8 h maintained cholecystokinin levels but did not alter cell growth. These data support roles for epidermal growth factor and insulin as growth factors for human pancreatic cancer cell lines.

Dexamethasone-induced killing of neoplastic cells of lymphoid derivation: lack of early calcium involvement.

The role of calcium influx in dexamethasone-induced fragmentation of DNA was studied in the glucocorticoid-sensitive human lymphoid line of T cell derivation (CEM-C7). Reduction of calcium content in the medium or the use of EGTA increased DNA fragmentation and appeared to slightly enhance the effect of dexamethasone. Incubation of isolated nuclei in the presence of high concentrations of calcium did not bring about significant DNA fragmentation. Calmidazolium, an antagonist of calmodulin dependent reactions did not reduce the sensitivity of CEM-C7 cells to dexamethasone nor did it modify the response to dexamethasone of the resistant CEM-C1 line. It appears that in contrast to rodent thymocytes, massive calcium influx is not per se responsible for the initiation of directed cell killing (apoptosis).

Comparison of dexamethasone and lovastatin (mevinolin) as growth inhibitors in cultures of T-cell derived human acute leukemia lines (CEM).

Because previous studies have shown that a reduction of cholesterol synthesis is one of the earliest effects of dexamethasone on neoplastic lymphoid cells, a study was made to compare dexamethasone to lovastatin, a specific inhibitor of cholesterol synthesis, which acts on 3-hydroxy-3-methylglutaryl coenzyme A reductase. Two cell lines were used, both derived from human acute T-cell leukemia, one dexamethasone-sensitive (CEM-C7), the other dexamethasone-resistant (CEM-Cl). The results revealed a similar pattern of resistance and sensitivity of both lines to lovastatin, although only the dexamethasone effect was reversed by 1 microM RU 486, the antiglucocorticoid steroid. The cell killing by dexamethasone and lovastatin had the characteristics of apoptosis.

Increased dolichol content in glucocorticoid-sensitive human T-cell leukemia line grown in the presence of dexamethasone.

Two cell lines, derived from human T-cell acute leukemia, one glucocorticoid sensitive (CEM-C7) and the other glucocorticoid resistant (CEM-C1) were grown in the presence of 1 x 10-7 M dexamethasone and were analyzed for their dolichol content. After 24 hrs of incubation, dolichols became significantly elevated in the sensitive but not in the resistant line. Lovastatin, the specific inhibitor of cholesterol synthesis did not affect dolichol levels in either of the two cell lines. The results raise the possibility that dolichol accumulation might be involved in the early stages of the glucocorticoid-induced apoptosis (directed cell killing).

Defective utilization of cholesterol esters from low-density lipoprotein in a human acute lymphoblastic leukemia T cell line.

The glucocorticoid sensitive CEM-C7 T cell line was derived from human acute lymphoblastic leukemia cells by Norman and Thompson (Cancer Res. 37 (1977) 3875). Madden et al. (Cancer Res 46 (1986) 617) have demonstrated that the growth inhibitory effect of glucocorticoids on these cells is due in part to an inhibition of cholesterol synthesis even in the presence of low-density lipoprotein (LDL)-containing serum. To delineate further the role of cholesterol in this growth inhibition, we have examined the ability of LDL-bound [3H]cholesterol linoleate to reverse the growth inhibitory effect of 1 microM dexamethasone on the CEM-C7 cells. LDL-bound cholesterol linoleate did not reverse the dexamethasone-mediated growth inhibition. Although incorporation of [14C]acetate into free cholesterol was inhibited only 20% by incubation with LDL, the presence of dexamethasone further inhibited acetate incorporation into free cholesterol in the LDL-treated cells. Dexamethasone had no effect on the uptake or utilization of LDL-bound cholesterol linoleate. Under all conditions, more than 99% of the acetate incorporated into cholesterol was present as free cholesterol, while over 80% of the LDL-derived cholesterol linoleate remained in the ester compartment. In contrast, in normal lymphocytes, over half the LDL-derived cholesterol was converted to free cholesterol. Direct analysis of the acid cholesterol ester hydrolase, the enzyme primarily responsible for processing LDL-bound cholesterol esters, revealed over 60-times the activity of this enzyme in the normal lymphocytes as compared to the activity present in the C7 cells. This finding cautions against the assumption that the presence of lipoprotein-containing serum provides an adequate, usable source of cholesterol for all cells.

The role of cholesterol in the glucocorticoid-mediated inhibition of cell cycle progression in human acute lymphoblastic leukemia cells.

Two glucocorticoid receptor-containing clones of human acute lymphoblastic leukemia, one (CEM-C7) sensitive and one (CEM-C1) resistant to dexamethasone (dex) were studied in an effort to identify the time course of the biochemical changes responsible for dex-induced growth inhibition of CEM-C7 cells. Cells were synchronized by treatment with 0.25 mM (C7) or 0.50 mM (C1) thymidine for 12 h followed by 0.025 micrograms/ml (C7) or 0.050 micrograms/ml (C1) colcemid for 12 h, then released either in the presence or absence of 1 microM dex. The inhibition of cellular proliferation which occurs at 48 h after release in the dex-treated CEM-C7 cells was preceded by an inhibition of acetate incorporation into cholesterol, first evident at 24 h, inhibition of protein synthesis at 30 h, and the development of a cell cycle block in G1 at 36 h. No inhibition of any of these parameters was seen in the resistant CEM-C1 cells. Thus the inhibition of cholesterol synthesis in the sensitive cells may be one of the earliest parameters affected by glucocorticoids.

Differential regulation of the synthesis of mannosylphosphoryl derivatives of dolichol and retinol in HeLa cells.

We have shown earlier that in HeLa S3G cells, glucocorticoids stimulate the synthesis of dolichyl phosphorylmannose (Dol-P-Man) with a concomitant increase in the glycosylation of proteins (Ramachandran, C.K., Gray, S.L. and Melnykovych G. (1982) Biochem. J. 208, 47-52). Although controversial, there have been several lines of evidence suggesting that the synthesis of retinyl phosphorylmannose (Ret-P-Man) and Dol-P-Man may be carried out by the same enzyme. We examined this possibility and conclude that in HeLa S3G cells the syntheses of Dol-P-Man and Ret-P-Man are catalyzed by two different enzymes located in the same microenvironment. Our conclusion is based on the following observations: exogenously added dolichyl phosphate and retinyl phosphate did not compete with each other; when the cells were grown in the presence of 1 microM dexamethasone, the microsomal synthesis of Dol-P-Man was stimulated, without affecting the Ret-P-Man synthesis; Arrhenius plots on Ret-P-Man and Dol-P-Man synthesis showed breaks at 22 and 37.7 degrees C.