George Melnykovych

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.

Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells☆

Abstract Studies designed to identify the site(s) of inhibition of cholesterogenesis by glucocorticoids in HeLa cells have been carried out. As reported earlier (Cavenee, W. K. and Melnykovych, G., 1977, J. Biol. Chem.252, 3272–3276), the increase in cholesterol synthesis from acetate after serum removal from the medium was partially suppressed by dexamethasone. However, this suppression was not accompanied by an expected decrease in activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34), the enzyme identified as the rate-limiting step in the biosynthetic sequence from acetate to cholesterol. Instead, HMG-CoA reductase activity became significantly higher in response to dexamethasone treatment. The present results show that dexamethasone-induced inhibition of sterol synthesis was not accompanied by any significant alteration in fatty acid synthesis. Acetyl-CoA synthetase (EC 6.2.1.1) and cytosolic acetoacetyl-CoA thiolase (EC 2.3.1.9) activities were also unchanged. In contrast, activity of cytosolic HMG-CoA synthase (EC 4.1.3.5) decreased in parallel to the inhibition of sterol synthesis by dexamethasone. A concentration-dependent inhibition of this enzyme by dexamethasone and a correlation with the 11β,17α,21-trihydroxy structure of C21 steroids were demonstrated. These results suggest that HMG-CoA synthase is controlling the rate of cholesterogenesis when cholesterol synthesis is partially suppressed by glucocorticoids.

Relationships between steroid binding and elevation of alkaline phosphatase in HeLa cells

Abstract 1. 1. Elevation of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) by prednisolone in growing cultures of HeLa-S3 cells was partially inhibited by several C21 steroids. The requirement necessary to bring about this effect included the 11-deoxy structure of the steroid. The reversal was observed also with those 11-hydroxysteroids which were inactive as alkaline phosphatase inducers because they lacked a hydroxyl in position 17 (e.g. corticosterone). In contrast, the 11-ketosteroids were inactive as inhibitors of alkaline phosphatase induction. The reduced derivatives of cortisol (e.g. tetrahydrocortisol) were similarly inactive. 2. 2. Ability to reverse elevation of alkaline phosphatase seemed to be related to the interference with binding of cortisol at the cell surface. This was evidenced by the lack of specific binding competition in cells detached from glass by treatment trypsin or by mechanical removal.

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.

Inhibition of cholesterol biosynthesis in HeLa cells by glucocorticoids

Abstract Dexamethasone, at concentrations ranging from 10 −6 M to 10 −8 M inhibited the incorporation of acetate into sterol fraction of S3G strain of HeLa cells. The effect was abolished by the presence of serum supplement in the culture medium. Dexamethasone had no effect on the incorporation of mevalonate or cholesterol itself, indicating possible involvement of HMG-CoA reductase. A survey of representative steroids of the C 21 series demonstrated a relationship between the structure required for glucocorticoid activity and the effect on cholesterol biosynthesis.

Alkaline phosphatase in HeLa cells: Stimulation by phospholipase A2 and lysophosphatidylcholine

Treatment of homogenates and plasma membrane preparations from HeLa cells with phospholipase A2 (EC 3.1.1.4) caused a 50% increase in activity of membrane-associated alkaline phosphatase. Lysophosphatidylcholine, dispersed in 0.15 M KCl, affected alkaline phosphatase in a similar fashion by releasing the enzyme from particulate fractions into the incubation medium and by elevating its specific activity. Higher concentrations of lysophosphatidylcholine solubilized additional protein from particulate fractions but did not further increase the specific activity of the released alkaline phosphatase. Particulate fractions from HeLa cells were exposed to the effects of liposomes prepared from lysophosphatidylcholine and cholesterol. The ratio of particulate protein/lysophosphatidylcholine (by weight) required for optimal activation of alkaline phosphatase was one. Kinetic studies indicated that phospholipase A2 and lysophosphatidylcholine enhanced the apparent V of the enzyme but did not significantly alter its apparent Km. The increased release of alkaline phosphatase from the particulate matrix by lysophosphatidylcholine was confirmed by disc electrophoresis. The release of the enzyme by either phospholipase A2 or by lysophosphatidylcholine appeared to be followed by the formation of micelles that contained lysophosphatidylcholine. The new complexes had relatively less cholesterol and more lysophosphatidylcholine than the native membranes. The possibility that lysophosphatidylcholine formed a lipoprotein complex with the solubilized alkaline phosphatase was indicated by a break point in the Arrhenius plot which was evident only in the lysophosphatidylcholine-solubilized enzyme but could not be demonstrated in alkaline phosphatase that had been released with 0.15 M KCl alone.

A photobleaching recovery study of glucocorticoid effects on lateral mobilities of a lipid analog in S3G HeLa cell membranes

Abstract Treatment of the S3G strain of HeLa cells with dexamethasone inhibits cholesterol synthesis and thus results in decreased plasma membrane cholesterol-to-protein ratios. Incubation of HeLa cells with dexamethasone for 24 h lowers the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) in intact cell plasma membranes and isolated plasma membranes (Johnston, D. and Melnykovych, G. (1980) Biochim. Biophys. Acta 596, 320–324). We have examined the effect of dexamethasone treatment of S3G HeLa cells on the lateral diffusion of the fluorescent lipid analogue 3,3′-dioctadecylindocarbocyanine iodide (DiI) by the fluorescence photobleaching recovery technique. The lateral diffusion of DiI was measured in cells 0, 2, 6, 12, and 24 h following treatment with dexamethasone and in cells identically handled without dexamethasone at 37°C. The diffusion constants of DiI in the treated and untreated cell membranes at zero time were (4.52±0.30) · 10 −9 cm 2 /s and (4.56±0.24) · 10 −9 cm 2 /s, respectively. There was no significant change in the lateral diffusion of DiI in the untreated cells over the 24 h period. The lateral diffusion of the lipid probe in the dexamethasone-treated cells began to increase 6 h following treatment and reached (6.43±0.27) · 10 −9 cm 2 /s at 24 h. The lateral diffusion of DiI was also measured at 25, 17, 10 and 4°C following 24 h incubation with and without dexamethasone. The effect of dexamethasone treatment on the lipid probe lateral diffusion observed at 37°C is decreased at 25°C and reversed in direction at 10 and 4°C. These results agree with those obtained in artificial systems containing varying amounts of cholesterol and support the suggestion that cholesterol acts to suppress phospholipid phase changes in animal cells. The lateral diffusion of DiI localized as a monolayer at a mineral oil-water interface was measured by fluorescence photobleaching recovery. The resulting data and the viscosity of the mineral oil were used to calculate the microviscosities of the plasma membranes of untreated and dexamethasone-treated cells at 25°C. Membrane microviscosities were also calculated from the fluorescence polarization studies cited above. In both cases the dexamethasone treatment reduced the apparent microviscosity by approximately 25%. However, the absolute microviscosity values obtained by the two techniques differ by a factor of 3.

Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme a synthase and the regulation of sterol synthesis in tissue culture cells

Abstract We have demonstrated previously (Ramachandran C.K., Gray S.L. and Melnykovych G. (1978) Arch. Biochem. Biophys. 189, 205–211) that in HeLa cells grown in presence of glucocorticoids, cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase (EC 4.1.3.5) rather than hydroxymethylglutaryl-CoA reductase changes coordinately with the rate of cholesterol biosynthesis. The present work represent studies of some of the characteristics of this enzyme and its response to conditions which modify cholesterogenesis. Addition of serum to the culture medium suppressed the hydroxymethylglutaryl-CoA synthase activity, and low density lipoprotein showed a higher inhibitory effect than high density lipoprotein. The presence of cholesterol, 7-keto-cholesterol and 25-hydroxycholesterol diminished the enzyme activity to a significant extent. However, freshly purified cholesterol added to the culture medium in the presence of vitamin E was not inhibitory. HeLa cell hydroxy-methylglutaryl-CoA synthase showed an optimum pH of 9 and a Km value of 295 μM. for acetyl-CoA. The enzyme was inhibited by acetoacetyl-CoA, while hydroxymethylglutaryl-CoA and hydroxymethylglutarate were not inhibitory. Studies with inhibitors of macromolecular synthesis showed that protein and RNA syntheses were necessary for the increase in hydroxymethylglutaryl-CoA synthase activity resulting from serum deprivation. In the presence of cycloheximide, an immediate fall away of the enzyme activity was observed. A t 1 2 for decay of approx. 20–22 h was calculated. The t 1 2 of this enzyme in dexamethasone-treated cells was about 12–14 h showing that dexamethasone may cause increased degradation of the enzyme. An investigation on the inhibitory effect of dexamethasone on hydroxymethylglutaryl-CoA synthase in several cell lines was also carried out. The result suggests that this enzyme coordinately changes with cholesterogenesis in a variety of cell lines when the synthesis of cholesterol is partially suppressed by dexamethasone.

Effects of cultural conditions on alkaline phosphatase and cell culture survival in the presence of prednisolone.

Abstract Addition of prednisolone (0.5 μg/ml) to two cell lines of putative human origin resulted in a higher level of alkaline phosphatase in one cell line (Henle embryonic intestine) but had the opposite effect on another cell line (HeLa S3) in which the enzyme level was suppressed by the steroid. Mechanical removal of cells from the glass surface during the preparation of inocula and a high cell to surface ratio during incubation favored high levels of alkaline phosphatase in both cell lines. In addition to its effect on alkaline phosphatase, prednisolone partially prevented leakage of ninhydrin positive substances from the cells and prolonged cell survival under a variety of sub-optimal growth conditions.

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.