Effect of ethanol on cell growth and cholesterol metabolism in cultured Hep G2 cells.

The Hep G2 human hepatoma cell line has been recognized as an excellent in vitro human model system. For this reason, this line was used to study the effect of ethanol on HMG-CoA reductase activity concerning cell growth and cholesterol metabolism. Cells were incubated in ethanol-containing medium (0-400 mmol/L) for up to 102 h. Ethanol caused an inhibition in the growth rate and in HMG-CoA reductase activity that could be reverted by the removal of ethanol from the culture medium, indicating no cellular damage. These changes cannot be ascribed to the regulatory effect of cholesterol levels, since its content was not modified either in the cells or in the medium. The addition of mevalonate to the culture medium could not revert the growth rate inhibition evoked by ethanol. Moreover, ethanol produced an increment in the cholesterol efflux in [3H]cholesterol-prelabeled cells. We conclude that the decrease in HMG-CoA reductase activity evoked by ethanol treatment on Hep G2 cells would not be the cause but the consequence of the impairment in cellular growth, since this impairment could not be reverted by the addition of mevalonate to the culture medium.     

Effects of accumulated S-adenosylmethionine on growth of yeast cells

S-adenosylmethionine (SAM) accumulated in cultured yeast cells and affected growth in two ways. High levels of intracellular SAM in yeast inhibited early growth, but increased growth in medium without sources of nitrogen and sulfur. Accumulated SAM in the yeast cells was recycled as a nutritional source depending on the sulfur and nitrogen contents of the medium.     

Effects of pH-variations on the kinetics of growth and energy metabolism in cultured T-lymphoma cells: a microcalorimetric study.

The progression of T-lymphoma cells (CCRF-CEM) growing in suspension has been monitored during long term (12-28 h) batch experiments using microcalorimetry. In parallel with the calorimetric measurements, changes in cell concentration, pH, p(O2) and concentrations of the main energy sources (glucose and glutamine) were determined. The overall metabolic rate per cell (as reflected by the heat production rate per cell, Pcell) and the growth rate decreased with time. These changes could be attributed solely to the decrease in pH of the medium until the total heat production, Q, exceeded 1.2 J per ml (corresponding to an incubation time of 20 h of a batch having an initial cell concentration of 1 x 10(6) cells per ml). The lowering of p(O2) to a level of 0.02 mmol/l or the decrease in concentrations of glucose and glutamine to 7.7 and 1.3 mmol/l, respectively, did not influence Pcell or the growth pattern. No "crowding effect" was observed for the cells in the investigated concentration range (0.6-1.3) x 10(6) cells per ml.     

Metabolism,cellular actions,and cytotoxicity of selenomethionine in cultured cells

Selenomethionine metabolism and the biochemical basis for its cytotoxicity were analyzed in cultured human and murine lymphoid cells. The metabolic pathways were also addressed, using purified mammalian enzymes and crude tissue extracts. Selenomethionine was found to be effectively metabolized to S-adenosylmethionine analog, and that analog was further metabolized in transmethylation reactions and in polyamine synthesis, similarly to the corresponding sulphur metabolites of methionine. Selenomethionine did not block these pathways, nor was there a specific block on the synthesis of DNA, RNA, or proteins when added to the culture medium. Selenomethionine showed cytotoxicity at above 40 microM levels. Yet, low selenomethionine levels (10 microM) could replace methionine and support cell growth in the absence of methionine. Selenomethionine toxicity took place concomitantly with changes in S-adenosylmethionine pools. D-form was less cytotoxic than L-form. Methionine concentration modified the cytotoxicity. Together, this indicates that selenomethionine uptake and enzymic metabolism are involved in the cytotoxicity in a yet unknown way.     

Effects of fowlpox virus infection on lipid metabolism in cultured chicken embryo cells.

Lipid biosynthesis was measured in cultured chicken embryo cells after infection with fowlpox virus. Between 24 and 72 h postinfection, fowlpox virus-infected cells incorporated less [14C]acetate and 3H2O into fatty acids and sterols than did mock-infected cells, demonstrating a virus-dependent inhibition of general lipid metabolism. Two specific effects of fowlpox virus infection were an accumulation of C-4 alkylated sterol intermediates and inhibition of monounsaturated fatty acid biosynthesis.     

Diethylglyoxal bis(guanylhydrazone), a potent inhibitor of mammalian S-adenosylmethionine decarboxylase. Effects on cell proliferation and polyamine metabolism in L1210 leukemia cells

The polyamines are cell constituents essential for growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in the polyamine biosynthetic pathway. Methylglyoxal bis(guanylhydrazone) (MGBG) is an anti-leukemic agent with a strong inhibitory effect against AdoMetDC. However, the lack of specificity limits the usefulness of MGBG. In the present report we have used an analog of MGBG, diethylglyoxal bis(guanylhydrazone) (DEGBG), with a much greater specificity and potency against AdoMetDC, to investigate the effects of AdoMetDC inhibition on cell proliferation and polyamine metabolism in mouse L1210 leukemia cells. DEGBG was shown to effectively inhibit AdoMetDC activity in exponentially growing L1210 cells. The inhibition of AdoMetDC was reflected in a marked decrease in the cellular concentrations of spermidine and spermine. The concentration of putrescine, on the other hand, was greatly increased. Treatment with DEGBG resulted in a compensatory increase in the synthesis of AdoMetDC demonstrating an efficient feedback control. Cells seeded in the presece of DEGBG ceased to grow after a lag period of 1–2 days, indicating that the cells contained an excess of polyamines which were sufficient for one or two cell cycles in the absence of polyamine synthesis. The present results indicate that analogs of MGBG, having a greater specificity against AdoMetDC, might be valuable for studies concerning polyamines and cell proliferation.     

S-adenosylmethionine synthetase in cultured normal and oncogenically-transformed human and rat cells

We have investigated the enzymatic formation of S-adenosylmethionine in extracts of a variety of normal and oncogenically-transformed human and rat cell lines which differ in their ability to grow in medium in which methionine is replaced by its immediate precursor homocysteine. We have localized the bulk of the S-adenosylmethionine synthetase activity to the post-mitochondrial supernatant. We show that in all cell lines a single kinetic species exists in a dialyzed extract with a Km for methionine of about 3-12 microM. In selected lines we have demonstrated a requirement for Mg2+ in addition to that needed to form the Mg X ATP complex for enzyme activity and have shown that the enzyme can be regulated by product feedback inhibition. Because we detect no differences in the enzymatic ability of these cell extracts to utilize methionine for S-adenosylmethionine formation in vitro, we suggest that the failure of oncogenically-transformed cell lines to grow in homocysteine medium may result from the decreased methionine pools in these cells or from the loss of ability of these cells to properly metabolize homocysteine, adenosine, or their cellular product S-adenosylhomocysteine.     

Compartmentalization of cholesterol metabolism and cellular growth in cultured intestinal crypt cells

Growth of rat intestinal crypt derived cells IEC-6 ceased when the key enzyme of cholesterol synthesis, hydroxymethylglutaryl-CoA reductase, was blocked by the competitive inhibitor mevinolin. This effect was reversed by the addition of mevalonolactone. LDL suppressed reductase activity as well as cholesterol synthesis from [14C]octanoate and stimulated acyl-CoA cholesterol acyltransferase, but failed to support cell growth despite rapid receptor mediated degradation even in the presence of low mevalonolactone concentrations. Inhibition of cholesterol esterification by Sandoz-Compound 58-035 enhanced cell growth in the presence of mevinolin, but did not promote proliferation in the additional presence of low-density lipoproteins. HDL3 but not HDL2 or tetranitromethane-modified HDL3 totally reversed the mevinolin induced inhibition of cell growth. This rescue by HDL3 was overcome by an increased dose of mevinolin. HDL3 derepressed reductase, stimulated cholesterol synthesis and reduced cholesterol esterification, but did not reverse the cholesterol synthesis inhibition by mevinolin. It is concluded that IEC-6 cells preferentially use endogenously synthesized cholesterol for membrane formation rather than low-density lipoprotein cholesterol. High-density lipoproteins appear to normalize cell growth in the presence of mevinolin by inhibition of cholesterol esterification and probably by inducing the formation of non sterol products of mevalonate.     

Effects of cortisol, 17beta-estradiol and thyroliberin on prolactin and growth hormone production, cell growth and cell cycle distribution in cultured rat pituitary tumour cells.

Prolactin and growth hormone production were measured in a rat pituitary tumour cell strain (GH₃) after treatment with cortisol (5 × 10^?6 M), thyroliberin (2.5 × 10^?6 M) and 17β-estradiol (10^?6 M). The changes in hormone production were related to alterations in cell growth rate and cell cycle distribution. Cortisol inhibited prolactin production, stimulated growth hormone production and reduced the cellular growth rate measured two days after start of treatment (maximum about 40% inhibition). Flow-micro fluorometric analysis of DNA distributions showed that cortisol treatment reduced the relative number of cells in S phase (maximum effect about 50%) with a compensatory increase of the proportion of cells in G₁ phase. The lack of inhibition of prolactin production after three days of cortisol treatment may partly be related to the increased number of cells in G₁ phase. Thyroliberin and 17β-estradiol did not significantly affect cell growth after six days of treatment, although the fraction of cells in S phase was reduced by approximately 40% with a corresponding increase of cells in G₁ phase. For thyroliberin and 17β-estradiol, the stimulatory effect on prolactin production and the inhibitory effect on growth hormone production witin a period of treatment of six days cannot be explained by a shift in cell cycle distributions. None of the three hormones influenced the growth fraction which was equal to unity. In conclusion, thyroliberin and 17β-etradiol are able to change prolactin and growth hormone production without altering the cell cycle distribution. However, the effects of cortisol on prolactin and growth hormone production may partly be due to an alteration in cell cycle traverse resulting in an increased number of cells in the G₁ phase.     

Effects of cell density and cell growth alterations on cyclic nucleotide levels in cultured human diploid fibroblasts.

cGMP and cAMP concentrations were studied in cultures of two strains of normal human diploid lung fibroblasts, WI38 and KL-2, under various conditions which alter growth rate. Higher levels of cAMP were found in fibroblasts grown in medium with low (0.1 – 1.0%) serum concentration and thus exhibiting a decreased rate of growth. A rise in cAMP also preceded the decreased growth rate when medium was not changed for 4 days or longer (starvation). The reinitiation of cell growth by addition of fresh medium containing the standard 10% serum to either starved or serum-restricted cells was preceded by a rapid drop in cAMP level. Cellular cAMP levels increased to a moderate extent as sparse cultures first increased in density, but did not continue to rise as the culture approached saturation density. cGMP levels were inversely related to cell density: much higher cellular cGMP levels were found at low density than at higher cell density, whether cells were rapidly proliferating under standard growth conditions or had their growth arrested by omission of medium change or restriction of serum. Thus, under these conditions the steady state levels of cGMP appear to be related to cell density rather than rate of cell proliferation. However, a transient but appreciable increase in cGMP did occur upon the addition of fresh medium containing 10% serum to starved or serum-restricted cells, a condition leading to reinitiation of cell proliferation. Smaller but significant increases in cGMP were also evident following routine addition of fresh medium with serum to growing cells fed every other day and following mild EDTA-trypsin treatment of confluent WI38 fibroblasts. Thus, at least dual control mechanisms appear to be involved in the regulation of cGMP levels. Comparison of mid- and late-passage WI38 cells revealed no significant differences either in the levels of cGMP at sparse densities or in the density-dependent change in levels. These results suggest that levels of both cAMP and cGMP are influenced by cell density and also by conditions which alter the rate of cell proliferation.     

Effects of myostatin and other growth factors on cultured human cells

A dedicated cell-based biological test system was used for studying specific effects of myostatin and other human growth factors on the proliferation of cultured myoblasts and fibroblasts. Myostatin inhibited myoblast growth without affecting human fibroblasts. In this test system, human growth hormone and insulin-like growth factor I acted as antagonists of myostatin, which indicates that these agents have a potential for blocking its effects in vivo.     

Effects of propylthiouracil and methylmercaptoimidazol on metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells.

Monkey hepatocarcinoma cell monolayer cultures (NCLP-6E) metabolized thyroxine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine and 3,3'-diiodothyronine by phenolic and nonphenolic ring deiodinations and sulfation of the deiodinated products, as shown in previous work with this system. The effects of the antithyroid drugs, propylthiouracil (PTU) and methylmercaptoimidazole (MMI), on these processes was investigated. PTU, at 0.1 and 1 mM, inhibited only phenolic ring deiodination. MMI at 1 mM had no effect, but 32 mM inhibited deiodination of both rings as well as sulfation. The findings suggest that the increased serum rT3 level caused by PTU in vivo is the result of decreased rT3 deiodination, in contrast to the increased rT3 production which is caused by starvation.     

Serum-independent human lymphoblastoid cells. Effects of cell density on growth

A serum-independent (SI) line of human lymphoblastoid cells has been developed from a clone of the serum-dependent (SD) line RPMI 8866. The SI cells have been growing in serum-free culture for more than one year. In high serum concentrations the growth of the SI cells is identical to that of the SD cells. At low serum concentrations the SD cells die while the SI cells survive and grow. The growth of SI cells is density-dependent and can be overcome by the addition of serum, conditioned medium, or daily feeding with fresh medium.     

Glucose transport and metabolism in cultured cells of nervous tissue.

The effect of the concentration of glucose in the medium on the intracellular concentrations of metabolites of C-6 astrocytoma cells and C-1300 neuroblastoma cells in culture has been investigated. The intracellular concentrations of glucose, glycogen, glucose 6-P and UDP-glucose were measured at intervals after feeding the cells. A rapid increase in glucose and glucose 6-P levels occurred when fresh medium containing 5.5 mM glucose was applied to the cells, followed by slower increases in UDP-glucose andglycogen. When the medium glucose was increased ten-fold, the intracellular concentration of glucose was increased, but the level of glucose 6-P, UDP=-glucose and glycogen were not altered, nor were the rates of accumulation. The addition of insulin to the medium resulted in an increase of intracellular glucose, glucose 6-P and glycogen. The transport of glucose into the cells is not the rate-limiting step of the regulation of metabolite levels in the cells.     

Cystine/cysteine metabolism in cultured Sf9 cells: influence of cell physiology on biosynthesis, amino acid uptake and growth

Spodoptera frugiperda (Sf9) insect cells proliferate in a cystine-free medium, with the same growth rate, reaching the same final cell density, as in a cystine-containing medium, provided that the inoculum is taken from a pre-culture sufficiently early, at 47–53 h. With an inoculum from a 103 h culture an extended lag phase accompanied by cell death was observed during the first 50 h of cystine-free culture, even though the culture had been adapted to cystine-free conditions for 10 passages. Cystine-free cultures seeded with a 103 h inoculum had lower growth rates and reached lower final cell densities than corresponding cystine-supplied cultures. Cysteine biosynthesis occurs from methionine via the β-cystathionine pathway. More methionine was consumed by the cells in cystine-free media, and cystathionine was secreted when methionine and cystine were supplied in excess. The data suggest that cysteine biosynthesis is up-regulated in proliferating cells but down-regulated when the cells enter the stationary phase. In cultures supplied with cystine (10–100 mg 1^-1), the specific uptake rate and total consumption of cystine, as well as the uptake of glutamate, glutamine and glucose increased with increasing cystine concentrations. These results are interpreted in view of system x _c ^– , a concentration dependent amino acid transporter. Similarly, the consumption of amino acids transported by system L (ile, leu, val, tyr) was enhanced in cystine-containing cultures, as compared to cystine-free cultures. Uptake of cystine, methionine and system L amino acids ceases abruptly in all cultures, even before growth ceased. The specific growth rate starts to decline early during the growth phase, but this growth behaviour could not be correlated to the depletion of nutrients. We therefore propose that the observed growth pattern is a result of (auto)regulatory events that control both proliferation and metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

Collagenolytic activities of cultured human malignant melanoma cells.

Three human malignant melanomas were cultured in pure populations and one tumor was cloned into melanotic and amelanotic cell lines. In the homogenates of these cultured cells, specific collagenase activities were demonstrated by isotope release from 14C-labeled collagen, disc electrophoresis, and specific cleavage of collagen molecules as demonstrated in the segment long spacing form. No significant collagenase activity was observed in the culture media. Interestingly, early cultures had a high collagenase activity in the cells and as they were successively subcultured, the activity diminished. Cysteine completely inhibited the degradation of tropocollagen as determined by disc electrophoresis and EDTA partially inhibited the degradation. It is concluded that human malignant melanoma cells produce a specific collagenase in vitro which can be extracted in early culture directly from the homogenate.