Control of 3-hydroxy-3-methylglutaryl coenzyme A reductase by endogenously synthesized sterols in vitro and in vivo.

Isolated rat hepatocytes converted mevalonolactone into sterol intermediates and fatty acids 6- to 8-fold faster than mevalonate salt at concentrations less than 6 X 10(-4) M. Incubation of hepatocytes for 3 h normally results in induction of 3-hydroxy-3-methylglutaryl-CoA reductase. This increase in enzyme activity was inhibited by mevalonolactone and by mevalonate salt; at each concentration between 6 X 10(-4) M and 6 X 10(-8) M the lactone was a more effective inhibitor than the salt. The increase in enzyme activity was completely prevented by 6 X 10(-4) M lactone, and at this concentration the cells synthesized from the lactone an amount of sterol per hour which approximated that leavingthe cells in the same period. Administration of mevalonolactone to intact rats resulted in a dose-dependent inhibition of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity. At the highest dose (400 mg of (RS)-mevalonolactone/200 g of rat) enzyme activities declined 85% within 45 min and were still suppressed below normals after 28 h. Mevalonolactone treatment resulted in increases in liver cholesterol content and in the cholesterol ester concentration of liver microsomes. The results demonstrate that the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase can be controlled by the rate of endogenous sterol synthesis both in vitro and in vivo.     

Synthesis of prostaglandin F by cultured human endometrial cells

Human endometrial cells were dispersed with collagenase and maintained in culture overnight. The synthesis of PGF by the dispersed cells incubated at 37 degrees C in serum-free medium was stimulated by estradiol (10(-7)M - 10(-5)M), histamine (5X10(-7)M - 5X10(-5)M), bradykinin (10(-6)M), phorbol myristate (PMA, 3X10(-8)M) and arachidonate (5X10(-6)M). Preincubation of the cells for 3 h with cortisol (5X10(-7)M - 5X10(-5)M), progesterone (10(-6)M) or mepacrine (10(-6)M - 2X10(-4)M) inhibited the response to histamine, bradykinin and PMA but not to arachidonate. Perfusion of the cultured cells in filtration chambers yielded similar results to those obtained in the incubation system but differences in the onset and duration of the responses to stimuli were found. In the perifusion system the responses to histamine and bradykinin were rapid and of short duration (peak response in less than 60 min) while the responses to PMA and arachidonate were of longer duration with a slower onset. We conclude that these observations using dispersed endometrial cells are consistent with previous work showing that histamine, bradykinin and PMA act by stimulating acylhydrolase activity, thereby liberating precursors such as arachidonic acid which are converted to prostaglandins by the cyclo-oxygenase complex.     

Tetrahydrouridine specifically facilitates deoxycytidine incorporation into herpes simplex virus DNA.

As reported by Jamieson and Subak-Sharpe (J. Gen. Virol. 31:303-313, 1976), exogenous deoxycytidine is very poorly incorporated into herpes simplex virus DNA. Here it is shown that this incorporation was dramatically increased in the presence of tetrahydrouridine (THU), a specific inhibitor of cytidine-deoxycytidine deaminase. Thus, the exclusion of deoxycytidine from herpes simplex virus DNA probably results from massive degradation by the deaminase, which is consistent with the observation that in the absence of THU, most of the nucleotides formed from exogenous deoxycytidine are dUMP. The effect of tHU upon deoxycytidine incorporation was specific for herpes simplex virus-infected cells; THU did not increase deoxycytidine incorporation into DNA of uninfected cells. Therefore, one might expect THU to enhance the antiviral activity of 1-beta-D-arabinofuranasylcytosine since this analog is also readily deaminated. However, THU increased both the antiviral activity and the cell toxicity only slightly and to about the same extent. Therefore, the metabolism of 1-beta-D-arabnofuranosylcytosine is different from that of deoxycytidine in herpes simplex virus-infected cells.     

Methylxanthine bronchodilators potentiate multiple human neutrophil functions

Methylxanthines, including the bronchodilators theophylline and aminophylline, in high concentrations (greater than 10(-4) M) inhibit cyclic nucleotide phosphodiesterase activity and in low, clinically relevant concentrations (10(-5) to 10(-4) M) are antagonists of extracellular adenosine receptors. The effect of therapeutic concentrations of methylxanthines on human neutrophil functions stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) was examined. Preincubation of cytochalasin B-treated neutrophils with 10(-5) M to 3 X 10(-3) M methylxanthine resulted in a biphasic, concentration-dependent effect on neutrophil aggregation, lysosomal enzyme release, and superoxide anion formation. At 10(-5) to 10(-4) M, theophylline and aminophylline potentiated neutrophil aggregation, lysosomal enzyme release (30 to 50%, p less than 0.005), and superoxide anion formation (30 to 60%, p less than 0.005). 1-Methyl-3-isobutylxanthine at these same concentrations potentiated only neutrophil aggregation and lysosomal enzyme release (30 to 40%, p less than 0.005). The three methylxanthines inhibited each response up to 90% at concentrations greater than 10(-4) M. 8-Phenyltheophylline, which does not inhibit phosphodiesterase activity, produced only potentiation. Preincubation of neutrophils with adenosine deaminase mimicked the methylxanthine potentiation, whereas addition of adenosine (3 X 10(-8) to 3 X 10(-7) M) reversed the methylxanthine-induced potentiation in a concentration-dependent manner. These results indicate that therapeutic concentrations of methylxanthines may potentiate neutrophil activation in vivo by competing with circulating adenosine for neutrophil adenosine receptors.     

Isolation and characterization of human liver guanine deaminase

Guanine deaminase (EC 3.5.4.3, guanine aminohydrolase [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a pI of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent Km for guanine was determined to be 1.53 X 10(-5) M at pH 6.0 and 2 X 10(-4) M for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a Ki of 1.53 X 10(-5) M and a Ki of 5 X 10(-5) M with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 X 10(-4) M and a 24% loss at 1 X 10(-3) M after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 X 10(-4) M. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 degrees C, with a loss of almost all activity at 65 degrees C with 30 min incubation. Two pKa values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine.     

Activation of a well behaved cell cycle in araC-treated V79 cells by caffeine

0.3–1.0 σmM araC (cytosine arabinoside) treatment of V79 cells produced inhibition of multiplication of cells which was accompanied by a large increase of cell size. In presence of 1–2 mM caffeine the inhibition of cell proliferation due to araC treatment was substantially reduced and cell-size increase was prevented; caffeine did not influence the uptake of araC by V79 cells. Flow microfluorometric analysis showed that caffeine induced a wave of cell cycle progression in 0.3 μM araC-treated cells. The cell cycle activated by caffeine in 0.3 μM araC-treated cells was largely well behaved; this was indicated by the fact that (1) prior to cell division cells achieved a tetraploid DNA content and (2) following cell division they had diploid DNA content as a result of which DNA homeostasis was maintained. At 1.0 μM araC concentration, however, extreme micronucleation was observed which gave rise to a substantial fraction of micronuclei with < G1 DNA content.     

A recombinant apoA-1--protein A hybrid reproduces the binding parameters of HDL to its receptor.

We have constructed a plasmid, pLM8, containing the coding sequence of the mature human apoA-1 fused to the coding sequence of the IgG-binding domains of protein A (PA) from Staphylococcus aureus. The hybrid gene is transcribed in Escherichia coli under the control of a heat-sensitive repressor, leading to the synthesis of large amounts of hybrid protein (apoA-1--PA). The hybrid protein was purified by denaturation with urea and alkali, renaturation and affinity chromatography on an IgG Sepharose column. ApoA-1--PA is soluble and has an Mr of 316 kd, as determined by gel filtration. This is five times the monomer size of 62 kd, predicted from the sequence and found by SDS-PAGE analysis. Cell surface binding activity of the hybrid protein was tested using two different cell types (J774 macrophages and Fao hepatocytes) and compared to human high density lipoprotein (HDL). High-affinity binding was found for both ligands in both cell lines (Kd = 3.4 X 10(-8)M in Fao cells, 4.9 X 10(-8) M in J774 cells for apoA-1--PA and 3.0 X 10(-8) M in Fao cells, 2.8 X 10(-8) M in J774 cells for HDL), with approximately 2 X 10(5) high-affinity binding sites per cell. ApoA-1--PA and HDL effectively competed with each other for binding to the cell surface. Additionally, they both bound to a 110-kd polypeptide on a ligand blot, identifying an HDL receptor. The binding parameters of HDL were very similar to those of apoA-1--PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation between DNA replication and neutral Mn 2+-dependent DNAse activity in chromatin

In cultures of primary murine fibroblasts the 10% serum stimulates the replicative synthesis of DNA inhibited by aphidicolin and araC (cytosine arabinoside). Using direct immunofluorescence analysis, it was shown that antibodies penetrate inside the cells and after 4 hours are pooled in the nuclei, where they remain for another 20 hours. The substitution of antibodies against chromatin DNAase by bovine serum albumin of normal serum gamma-globulins does not interfere with the DNA synthesis induction.     

Norepinephrine-stimulated vascular prostacyclin synthesis. Receptor-dependent calcium channels control prostaglandin synthesis

Norepinephrine-stimulated prostacyclin synthesis was studied in rat aortic rings by measuring 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay. Norepinephrine (10(-6) M) results in a 10- to 20-fold increase in 6-keto-PGF1 alpha synthesis by rat aortic rings (54 +/- 11 to 437 +/- 260 pg X mg wet weight-1 X 20 min-1). The maximal stimulation of 6-keto-PGF1 alpha synthesis was observed with a norepinephrine concentration of 10(-5) M at a mean effective concentration (EC50) of 9.5 +/- 3.2 X 10(-7) M which is similar to the contractile response (Emax = 10(-5) M, EC50 = 6.5 +/- 1.8 X 10(-7) M). Potassium chloride (30 mM), although causing a similar maximal contractile response as 10(-6) M norepinephrine, did not increase 6-keto-PGF1 alpha synthesis. Norepinephrine-stimulated 6-keto-PGF1 alpha synthesis was dependent upon extracellular calcium. Norepinephrine stimulation in Ca2+-free medium did not lead to a significant increase in 6-keto-PGF1 alpha synthesis. However, on the introduction of Ca2+, 6-keto-PGF1 alpha synthesis was restored to its initial level. Phentolamine (10(-6) M) (an alpha-adrenergic antagonist) and trifluroperazine (2.5 X 10(-4) M) (a calmodulin inhibitor) completely inhibited norepinephrine-stimulated 6-keto-PGF1 alpha synthesis, whereas verapamil 3 X 10(-6) M (a calcium channel blocking drug) only partially inhibited synthesis (control, 74 +/- 12; norepinephrine, 437 +/- 260; norepinephrine + verapamil, 123 +/- 8 pg X mg wet weight-1 X 20 min-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the monovalent ionophore monensin on the intracellular transport and processing of pro-opiomelanocortin in cultured intermediate lobe cells of the rat pituitary

Detailed studies on the effects of the ionophore monensin upon synthesis, maturation, and intracellular transport of pro-opiomelanocortin in cultures of rat pituitary intermediate lobe cells have been carried out. When added at concentrations larger than 5 X 10(-8) M monensin significantly inhibited protein synthesis by cultured intermediate lobe cells. Pro-opiomelanocortin synthesis was also reduced proportionally to the overall rate of protein synthesis. During pulse-chase experiments, monensin when added at a concentration of 10(-5) M at the beginning of the chase incubation completely inhibited the proteolytic processing of pro-opiomelanocortin. Using a subcellular fractionation procedure of intermediate lobe cell extracts on Percoll gradients, we were able to show that after the addition of monensin (10(-5) M), labeled pro-opiomelanocortin molecules synthesized during a 15-min pulse-incubation were recovered intact after a 2-h chase, in the fractions of the density gradient corresponding to the rough endoplasmic reticulum and Golgi elements. No maturation products or precursor molecules entered the granule fractions as observed in nontreated cells. Taken together these results strongly suggest that monensin blocks the intracellular transport of newly synthesized pro-opiomelanocortin molecules at the Golgi level and that inhibition of proteolytic processing is due to the failure of the prohormone to enter the cell compartment (probably the secretion granules) where maturation proteases are located.     

Mineralocorticoid-specificity of aldosterone-induced protein synthesis in giant-toad (Bufo marinus) urinary bladders.

We have identified a group of proteins (Mr approximately 70000-80000; pI approximately 5.8-6.4) in giant-toad (Bufo marinus) urinary-bladder epithelial cells whose synthesis appears to be related to aldosterone-stimulated Na+ transport. To define this relationship further, we examined whether submaximal natriferic concentrations of aldosterone induced these proteins and whether spironolactone (a specific mineralocorticoid antagonist in renal epithelia) inhibited their synthesis. Short-circuit current was used to measure Na+ transport and epithelial-cell protein synthesis was detected with high-resolution two-dimensional polyacrylamide-gel electrophoresis and autoradiography. Submaximal natriferic concentrations of aldosterone (1.4 X 10(-8) M) induced the same proteins as maximal concentrations of the hormone (1.4 X 10(-7) M). In contrast, in previous experiments, similar proteins were not induced by subnatriferic concentrations (5.0 X 10(-8) M) of cortisol, a glucocorticoid. A spironolactone/aldosterone molar ratio of 2000:1 was required to inhibit aldosterone-stimulated Na+ transport completely; ratios of 200:1 and 500:1 produced partial inhibition. Concentrations of spironolactone that abolished aldosterone-stimulated Na+ transport also inhibited aldosterone-induced protein synthesis. We conclude that the synthesis of the proteins we have identified is specifically related to activation of the mineralocorticoid pathway.     

Increase of (Ca2++Mg2+)-ATPase activity of renal basolateral membrane by parathyroid hormone via cyclic AMP-dependent membrane phosphorylation

Studies were made on the mechanism of the effect of parathyroid hormone (PTH) on the activity of (Ca2++Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme from the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.3 X 10(-7) M, Vmax = 200 nmol Pi/mg/min). At 1 X 10(-7) M free Ca2+, both PTH (10(-7)-10(-6) M) and cAMP (10(-6)-10(-4) M) stimulated (Ca2++Mg2+)-ATPase activity dose-dependent and their stimulatory effects were inhibited completely by 5 microM H-8, an inhibitor of cAMP-dependent protein kinase. PTH (10(-7) M) also caused 40% increase in 32P incorporation into the BLM and 5 microM H-8 inhibited this increase too. PTH (10(-7) M) was found to stimulate phosphorylation of a protein of Mr 9000 by cAMP dependent protein kinase and 5 microM H-8 was found to block this stimulation also. From these results, it is proposed that PTH stimulates (Ca2++Mg2+)-ATPase activity by enhancing its affinity for free Ca2+ via cAMP-dependent phosphorylation of a BLM protein of Mr 9000.     

Sodium-dependent phosphate transport inhibited by parathyroid hormone and cyclic AMP stimulation in an opossum kidney cell line

In the present study we investigated the characteristics of the transport of inorganic phosphate (Pi) in an opossum kidney cell line endowed with parathyroid hormone (PTH) receptors. In confluent epithelial cell culture, a Na-dependent Pi transport (NaPiT) was identified. Preincubation for 1 h with bovine (b)PTH(1-34) at 10(-7) M inhibited the NaPiT from 2.76 +/- 0.11 to 1.08 +/- 0.10 nmol/mg protein X 2 min-1 (p less than 0.001). This inhibition was already expressed 5 min after exposure to 10(-7) M bPTH. It was associated with a 4-fold increase in cellular cyclic AMP. The NaPiT was significantly inhibited at 10(-9) M bPTH, a hormonal concentration which stimulated the cellular cyclic AMP by only 30%. Kinetic analysis of the NaPiT inhibition by 10(-7) M bPTH revealed a decrease in Vmax (from 4.14 +/- 0.32 to 2.41 +/- 0.14 nmol/mg protein X 2 min-1) with no change in Km (0.093 +/- 0.016 versus 0.094 +/- 0.012 mM). The effect of bPTH on NaPiT was not associated with a change in the Na-dependent glucose methylglucopyranoside transport also present in the opossum kidney cell line. The inhibitory influence of bPTH on NaPiT was not affected by blockage of new protein synthesis by cycloheximide. Stimulation of cyclic AMP production by 10(-5) M forskolin, 10 micrograms/ml cholera toxin, 10(-5) M prostaglandin E2 or addition of 10(-5) M dibutyryl cyclic AMP mimicked the PTH-induced reduction in NaPiT. In conclusion, the present study indicates that the opossum epithelial cell line is endowed with a Na-dependent Pi transport system which is selectively inhibited by PTH and agents which increase cyclic AMP production.     

Prostaglandins and the rat seminal vesicle: effects of mating and adrenergic stimulation

The concentrations of PGE, PGF, and 6-keto-PGF1 alpha were increased in rat seminal vesicle tissue following mating activity. Likewise, synthesis of PGE and PGF was stimulated by epinephrine (3 X 10(-7) to 3 X 10(-6) M) in tissues and media from in vitro incubations of intact rat seminal vesicles. The in vitro stimulation was inhibited by phentolamine, an alpha-adrenoreceptor blocking agent. Carbamylcholine (2 X 10(-6) M) and bradykinin (1 X 10(-6) M) had no effect on PGE or PGF synthesis, even though both compounds stimulated contractility of the rat seminal vesicle at these concentrations. These data suggest that mating and adrenergic stimulation increase prostaglandin synthesis in the rat seminal vesicle, probably through an alpha-adrenergically mediated mechanism.     

Enhancement of melanotic expression in cultured mouse melanoma cells by retinoids

Retinoic acid (RA), which reduces the rate of cell proliferation in S91 mouse melanoma clone C2 cells, was found to stimulate the expression of their melanotic phenotype. RA treatment also induced the extension of long cellular processes. The RA effects on melanogenesis included stimulation of tyrosinase activity and augmentation of cellular melanin content to levels 3- to 4-fold higher than in untreated cultures at similar cell densities. These effects became apparent after 48 hours of exposure to 10(-5) M RA and increased thereafter. Half-maximal stimulation in cells treated for 6 days occurred at 5 X 10(-7) M RA. Although the degrees of melanogenesis enhancement by RA (10(-5) M) and by alpha-melanocyte stimulatory hormone (2 X 10(-7) M) were similar, the former did not alter the intracellular cAMP level, whereas the latter induced a transient 4-fold increase. In high-passage (p28) cells, as well as in low-passage cells (less than p10) treated with tyrosinase inhibitor phenylthiocarbamate, melanin synthesis was suppressed in the absence and presence of RA, yet the ability of RA to inhibit cell proliferation was not compromised. In the presence of the tumor promotor phorbol myristate acetate (greater than 5 X 10(-9) M) melanin synthesis in control as well as in cells exposed to RA was dramatically inhibited. Phorbol which is not active in tumor promotion had no effect on melanogenesis. In addition to RA, other retinoids, such as 13-cis-retinoic acid, retinyl acetate, the TMMP analog of RA and the phenyl analog of RA, but not the pyridyl analog of RA or retinyl palmitate, also inhibited cell growth and enhanced melanin synthesis.     

Chemical synthesis of DNA oligomers containing cytosine arabinoside.

The solid phase phospite triester synthesis of oligodeoxynucleotides containing cytosine arabinoside (araC) is described. A protected araC phosphoramadite was prepared for the introduction of araC residues at 5'termini and internucleotide positions in DNA oligomers. These oligomers were utilized to demonstrate the formation of correct 3'-5' linkages, to test for alkaline lability at the araC site, and to study the stability of duplexes containing araC-G base pairs. For the introduction of araC residues at 3' terminal positions, a protected derivative of araC was coupled to functionalized silica. This material was used to prepare a test oligomer which was characterized enzymatically.     

Neplanocin A. A cyclopentenyl analog of adenosine with specificity for inhibiting RNA methylation

The mechanism of action of the adenosine analog, neplanocin A (NPC), was investigated in human colon carcinoma cell line HT-29. Cell viability was reduced to 38 and 17% of control by 24-h exposure to 10(-5) and 10(-4) M NPC, respectively. Cytocidal activity was not affected by inhibition of adenosine deaminase with 2'-deoxycoformycin. Concomitant with decreased cell viability was the reduced incorporation of [14C]dThd and [3H]Leu, and to a lesser extent [3H]Urd, into acid-precipitable material. Labeling of rRNA and tRNA during drug treatment for 24 h with [methyl-3H]Met and [14C]Urd revealed that NPC primarily inhibited RNA methylation, and to a lesser extent, RNA synthesis. RNase T2 digests of total RNA indicated that base and 2'-O-methylation were inhibited to approximately the same degree. Metabolites of NPC were measured by reverse-phase high-performance liquid chromatography and it was found that the major drug metabolite was the drug analog of S-adenosylmethionine with little formation of the respective, S-adenosylhomocysteine metabolite. NPC was utilized to a very small degree for RNA synthesis where only 2 and 30 pmol of NPC/A260 were incorporated into rRNA and tRNA after 24-h exposure to 10(-5) and 10(-4) M NPC, respectively. These results indicate that NPC is metabolized to a metabolite of S-adenosylmethionine which is a poor methyl donor for RNA methyltransferases, and that the accompanying decrease in RNA methylation and protein synthesis appears to be related to its cytocidal activity.     

Biphasic Modulation by ACTH-Like Peptides of Protein Synthesis in a Cell-Free System from Rat Brain

Brain protein synthesis in a cell-free system was stimulated by 10(-8) M-ACTH1-24. This stimulatory effect was completely inhibited by aurintricarboxylic acid (ATA), an inhibitor of reinitiation of new peptide chains. The N-terminal peptide sequence 4-10 exerted a biphasic modulation of cell-free protein synthesis, i.e., a stimulation at low concentrations (10(-8) and 10(-10) M) and an inhibition at a high concentration (10(-4) M). The D-isomer, ACTH4-10-7-D-phe, also showed a biphasic modulation that, however, was in a direction opposite to that shown by ACTH4-10-7-L-phe at 10(-8) M and 10(-4) M.     

Introduction of a 16 alpha-hydroxyl function into estrone by Streptomyces roseochromogenes

Streptomyces roseochromogenes (NRRL B-1233) converted estrone predominantly into its 16 alpha-hydroxyl derivative. Chemical and spectroscopic (UV, IR, NMR, MS) methods were used in establishing the structure and strereochemistry of the oxidation product. The product was assigned as 16 alpha-hydroxyestrone (yield, 17%). No other oxidation product was detected in this experiment. An interrelationship between cell growth and 16 alpha-hydroxy-estrone formation was observed. Also, 16 alpha-hydroxylation of estrone was observed in resting cells. 16 alpha-Hydroxylase showed good activity at 3.7 X 10(-4)M of estrone concentration and was completely inhibited by 1.1 X 10(-3)M. These results indicate the presence of a constitutive 16 alpha-hydroxylase in the organism investigated.     

Activation of a well-behaved cell cycle in araC-treated V79 cells by caffeine

.3-1.0 microM araC (cytosine arabinoside) treatment of V79 cells produced inhibition of multiplication of cells which was accompanied by a large increase of cell size. In presence of 1-2 mM caffeine the inhibition of cell proliferation due to araC treatment was substantially reduced and cell-size increase was prevented; caffeine did not influence the uptake of araC by V79 cells. Flow microfluorometric analysis showed that caffeine induced a wave of cell cycle progression in 0.3 microM araC-treated cells. The cell cycle activated by caffeine in 0.3 microM araC-treated cells was largely well behaved; this was indicated by the fact that (1) prior to cell division cells achieved a tetraploid DNA content and (2) following cell division they had diploid DNA content as a result of which DNA homeostasis was maintained. At 1.0 microM araC concentration, however, extreme micronucleation was observed which gave rise to a substantial fraction of micronuclei with less than G1 DNA content.