Effects of selective denervation and nerve growth factor on activity-mediated growth of rat parotid gland.

A dietary change from all liquid to solid food is followed by an average increase of 200% in [3H]thymidine uptake into the parotid gland of rat. However, removal of either the parasympathetic (Px) or the sympathetic (Sx) innervation to the parotid gland prior to the dietary change resulted in a partial inhibition of the increase; values for the parasympathectomized gland were 51% of those of the innervated gland, and values of the sympathectomized parotid gland were 42% of those of the innervated gland. Removal of both autonomic nerves caused a complete inhibition. Initiation of nerve growth factor (NGF) injection (1 microgram/kg body wt, two times daily for the 2 days of chow refeeding) at the time of chow refeeding had no effect on completely or partially denervated glands, and thymidine values for the denervated parotid gland of rats given NGF did not differ statistically from those of rats not given NGF. With parasympathectomy, sympathectomy, and complete denervation, weight of parotid gland was decreased from that of innervated glands, and administration of NGF had no effect on the denervation-induced decreases. The data show that both branches of the innervation to parotid gland must be intact to ensure a maximal increase in thymidine uptake with the dietary change from liquid to solid food. The level of the enzyme, beta 1-4 galactosyltransferase, involved in proliferation, also depended on the presence of intact nerves. Enzyme activity of innervated parotid gland showed an average increase of 200% with chow refeeding of rats previously on liquid diet, but with Px, the average increase was 51% of that of the innervated parotid, and with Sx, it was 41%. NGF administration did not cause any change in levels of this enzyme in any Px or PxSx parotid gland and only a small change in Sx parotid; it did increase levels of this enzyme in parotid of rats without submandibular-sublingual glands.     

Calcium concentration of saliva and salivary glands of rat after cyclocytidine

Cyclocytidine (CC), in addition to its antitumor properties, also causes copious flow of saliva. Calcium concentration of CC-evoked saliva from submaxillary (SM) and parotid (PA) glands of adult rats was initially 7 meq/liter and 15 meq/liter, respectively, and thus resembled that of sympathetically evoked secretion. From previous data, as well as present data, this is expected since CC apparently causes release of norepinephrine (NE) from adrenergic nerve endings. Present data also confirm that CC causes NE release since a single dose of reserpine (RES) (5 mg/kg), administered 24 hr prior to injection of CC in order to cause depletion of NE prevented the action of CC. Furthermore, the NE released by CC acts principally on beta-adrenergic receptors since propranolol administered prior to CC caused a marked reduction in flow and [Ca] of saliva, and prevented the usual CC-induced depletion of glandular calcium. An increase in [Ca] of SM but not PA gland was also caused by chronic (daily injections of 500 mg/kg body wt for 3 days) administration of CC. The same threefold increase was observed 2 days after injection of a single dose of CC also. The increase in glandular calcium was not prevented by propranolol, thus suggesting that this effect of CC on glandular [Ca] was probably not beta-mediated. The calcium increase may, however, be the result of depletion of NE. Thus, [Ca] of SM of CC-treated rats, that of RES-treated rats, and that of rats treated with RES + CC were very similar. If the mechanism of action of the two drugs were different (not NE depletion), the combined action of the two would have been additive.     

Initiation of DNA synthesis in murine B cells is independent of early changes in the cytosolic free calcium

The relationship between changes in the intracellular free Ca2+ concentration, [Ca2+]i, and the initiation of proliferation of murine B cells after the addition of mitogens and activators was studied. The effects of lipopolysaccharide (LPS), 12-O-tetradecanoyl phorbol-13-acetate (TPA), rabbit IgG antimouse Fab (IgG RAM Fab), and its F(ab')2 fragment (F(ab')2 anti-Fab) on the [Ca2+]i were measured using the fluorescent calcium indicator Fura-2. In parallel experiments, DNA and/or RNA synthesis were measured by assaying [3H]thymidine and/or [3H]uridine uptake. LPS stimulated a 20-120 X increase in the [3H]thymidine uptake, and a 3-7 X increase in [3H]uridine uptake without inducing any change in the [Ca2+]i. TPA induced a marginal increase in [3H]thymidine and [3H]uridine uptake, without effecting any change in the [Ca2+]i. In contrast, low doses of IgG RAM Fab produced a triphasic change in the [Ca2+]i, but had no effect on the [3H]thymidine or [3H]uridine uptake, even at much higher concentrations. Similarly, low doses of the F(ab')2 fragment induced sizable increases in the [Ca2+]i without affecting the [3H]nucleoside uptake. However, higher concentrations of F(ab')2 anti Fab increased the [3H]thymidine uptake and [3H]uridine uptake, while also increasing the [Ca2+]i. Significantly, pretreating the cells with TPA for 3 min virtually abolished the [Ca2+]i increase induced by IgG RAM Fab while simultaneously potentiating an increase in the IgG RAM Fab-induced [3H]thymidine uptake 85-fold. In the presence of TPA, IgG RAM Fab also induced a 2- to 30-fold increase in [3H]uridine uptake. Similarly, TPA virtually abolished the [Ca2+]i increase induced by the F(ab')2 anti-Fab fragment, yet it stimulated a F(ab')2 anti-Fab-induced uptake of [3H]thymidine and [3H]uridine by 120 and 10 times, respectively.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of (3H] uridine incorporation into RNA and [3H] leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10-21 M). Insulin stimulated the rate of [3H] thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100-1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [3H] thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of 3H- uridine, [3H] thymidine and [3H] leucine into their respective precursor pools is not responsible for the apparent stimulation of RNA, DNA and protein synthesis.     

Comparative developmental analysis of the parotid, submandibular and sublingual glands in the neonatal rat.

Analysis of the soluble protein fractions from the rat parotid, submandibular and sublingual glands by polyacrylamide-gel electrophoresis reveals similarities in overall patterns of protein synthesis at birth. Tissue-specific changes in protein and glycoprotein synthesis occur shortly after birth and again at the time of weaning, 21--28 days later. Incorporation of [3H]thymidine into DNA was at its highest after birth and gradually decreased in both the parotid and submandibular gland, whereas [3H]thymidine incorporation in the sublingual gland was low throughout the time of neonatal development. [14C]Leucine incorporation into total protein increased in all glands with age after birth, showing an accelerated rate 21--28 days later. Trichloroacetic acid/phosphotungstic acid-precipitable [3H]fucose in glycoproteins declined over the time of neonatal development in the parotid and submandibular gland, but its incorporation remained higher in the sublingual gland. alpha-Amylase (EC 3.2.1.1) in the salivary glands increased at the time of weaning, as judged by detectability in sodium dodecyl sulphate/polyacrylamide gels and by immune precipitation. Two membrane-bound enzymes, UDP-galactose:2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase (EC 2.4.1.22) and UDP-galactose:2-acetamido-2-deoxy-D-galactosaminyl-protein 3 beta-galactosyltransferase (no EC number), undergo tissue-specific change rather than changes induced by physiological stimulation of the salivary glands.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

The effect of pilocarpine and food uptake on the rate of incorporation of [3H]-leucine in vivo was measured by means of quantitative radioautography in three exocrine cells of the rat: the acinar and the granular duct cells of the submandibular and the acinar cells of the parotid gland. The three cell types react differently. The submandibular acinar cells showed a decrease in incorporation rate after pilocarpine administration but not after feeding. The incorporation rate of the granular duct cells of the submandibular gland remains constant after both stimulations. The acinar cells of the parotid gland show an increase in incorporation rate of [3H]-leucine in response to both. The contrast between the submandibular and the parotid gland could also be demonstrated radiobiochemically, the results reflecting the incorporation rates of the acinar cells of both glands, giving no information on the contribution of other cell types. The decrease in incorporation rate of the submandibular gland acinar cells is accompained by a shift of polyribosomes towards monomers.     

Down-regulation of cellular proto-oncogenes during inhibition of rat parotid acinar cell proliferation.

The role of cell surface galactosyltransferase in mediating isoproterenol-induced parotid gland hypertrophy and hyperplasia was examined in rat parotid gland acinar cells. Introduction of the transferase modifier, alpha-lactalbumin, or galactosyltransferase-associated kinase inhibitor trifluoperazine, into beta-agonist-treated rats prevented acinar cell proliferation as determined by [3H]thymidine incorporation after 96 h of treatment. However, [3H]thymidine incorporation into DNA after 24 h of treatment, with injection of a combination of isoproterenol/alpha-lactalbumin or isoproterenol/trifluoperazine, was similar to injections of isoproterenol alone; suggesting that acinar cells could be stimulated to undergo a single round of DNA synthesis. Northern blot analysis of myc and fos expression followed a similar pattern of down-regulation to control levels after 96 h but not after 24 h. Hybridization with erb B showed little change with proliferation, confirming previous observations on protein levels of the EGF-receptor in acinar cells. Western blot analysis of nuclear protein expression of myc revealed that isoproterenol caused an increase in a 62-kDa protein which was again down-regulated with inhibition of cell proliferation. Analysis of protein levels of Rb110 protein showed no change in protein level in the nucleus with cell proliferation, but did show an associated increase in protein phosphorylation in response to growth stimulation.     

Evidence for receptor-linked activation of phospholipase D in rat parotid glands. Stimulation by carbamylcholine, PMA and calcium.

In order to test if phospholipase D (PLD) activity exists in the rat parotid gland, we took advantage of the fact that, in the presence of ethanol, PLD generates phosphatidylethanol (PEth) via a transphosphatidylation reaction. Lipid extracts of parotid acini prelabelled with [3H]myristic acid were analyzed by thin layer chromatography to determine [3H]phosphatidylethanol ([3H]PEth) formation. Carbamylcholine (1 mM) stimulated [3H]PEth formation in the presence of 2% ethanol, this effect was completely inhibited by atropine (10 microM). PMA (0.1-1 microM) and ionomycine (10 microM) also caused [3H]PEth generation. We conclude that a phospholipase D activity is present in the rat parotid gland and is regulated by muscarinic cholinergic receptors. Protein kinase C and calcium could also modulate this activity. This report provides the first evidence for the existence and receptor-linked regulation of phospholipase D in an exocrine gland, the rat parotid gland.     

Metabolism of inositol phosphates in parotid cells: implications for the pathway of the phosphoinositide effect and for the possible messenger role of inositol trisphosphate

Rat parotid acinar cells were used to investigate the time course of formation and breakdown of inositol phosphates in response to receptor-active agents. In cells preincubated with [3H]inositol and in the presence of 10 mM LiCl (which blocks hydrolysis of inositol phosphate), methacholine (10(-4)M) caused a substantial increase in cellular content of [3H]inositol phosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Subsequent addition of atropine (10(-4) M) caused breakdown of [3H]inositol trisphosphate and [3H]inositol bisphosphate and little change in accumulated [3H]inositol phosphate. The data could be fit to a model whereby inositol trisphosphate and inositol bisphosphate are formed from phosphodiesteratic breakdown of phosphatidylinositol bisphosphate and phosphatidylinositol phosphate respectively, and inositol phosphate is formed from hydrolysis of inositol bisphosphate rather than from phosphatidyl-inositol. Consistent with this model was the finding that [3H]inositol trisphosphate and [3H]inositol bisphosphate levels were substantially increased in 5 sec while an increase in [3H]inositol phosphate was barely detectable at 60 sec. These results indicate that in the parotid gland the phosphoinositide cycle is activated primarily by phosphodiesteratic breakdown of the polyphosphoinositides rather than phosphatidyl-inositol. Also, the results show that formation of inositol trisphosphate is probably sufficiently rapid for it to act as a second messenger signalling internal Ca2+ release in this tissue.     

Inhibition of isoproterenol-induced DNA and RNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

Summary [³H] thymidine incorporation into DNA of the parotid (PA) gland of adult and 20-day-old rats and into DNA of the pancreas (PANC) of 20-day-old rats was increased markedly following a 2-day regimen of isoproterenol (ISO) administration. However, when the submandibular-sublingual (SM-SL) glands had been removed just prior to initiation of the ISO injections, the [³H] thymidine incorporation into PA and PANC was inhibited, and cpm/mg protein of these organs was even lower than that of organs of untreated rats with SM-SL glands present. Removal of the PA glands just prior to initiation of the ISO regimen had no effect on the ISO-induced [³H] thymidine incorporation into DNA of PANC but partially inhibited that of the submandibular (SM) gland. It is suggested that the inhibitory effects on DNA and RNA synthesis that follow removal of SM-SL glands are attributable to the growth factors (epidermal growth factor and nerve growth factor) found in the rat SM gland. These factors appear to regulate normal DNA synthetic activity of exocrine glands as well as ₁-adrenoceptor mediated DNA synthesis. Cellular hypertrophy induced by the ISO was less markedly affected by absence of the SM glands, but a partial inhibition of [³H] uridine incorporation into RNA of PA of adult rats also occurred when SM-SL glands were removed prior to initiation of the ISO-regimen.     

Purification of proline-rich proteins from parotid glands of isoproterenol-treated rats.

Prolonged isoproterenol treatment of rats is known to cause hypertrophy and hyperplasia of the parotid glands. Our results show that a dramatic increase in the synthesis or accumulation in the parotid glands of a series of proteins rich in proline also occurs with isoproterenol treatment. After 10 days of treatment (5 mg of isoproterenol/day) these proline-rich proteins (PRPs) comprise more than 50% of the total soluble proteins in parotid gland homogenates. The PRPs are rapidly labeled in vivo by a single intraperitoneal injection of [3H]proline with maximum incorporation occurring at about 3. More than 90% of the [3h]proline found in parotid gland homogenates is incorporated into PRPs with less than 1% of the radioactivity in alpha-amylase. Tritium incorporated into PRPs was isolated as [3H]proline after acid hydrolysis. One acidic and six basic 3H-labeled PRPs were isolated from the 100,000 x g supernatant fraction of parotid gland homogenates by Sephadex G-100 and ion exchange chromatography. The six basic proteins accounted for about 90% of the total PRPs isolated.     

Comparison of effects of surgical and chemical sympathectomy on beta adrenergic and muscarinic receptors of parotid gland of young and adult rats

Surgical (removal of a superior cervical ganglion) or chemical [(administration of a single dose of 6 hydroxydopamine (6 OHDA) (50 mg/kg dose body wt)] sympathectomy of rats at 2 or 8 days of age resulted in an increase in [3H]DHA binding of membranes of parotid gland of young rats (age range 21 days to 48 days). The increase progressed with postnatal age; at 21 days of age (surgical sympathectomy), it was 13%; at 32 days of age with 6 OHDA, it was as much as 34%, but only 26% at 42 days of age with surgical sympathectomy. No change in [3H]QNB binding was observed at any postnatal ages following neonatal sympathectomy. Conversely, surgical sympathectomy of the parotid of adult rat resulted in little or no change in [3H]DHA binding at 1, 2, 3, or 4 weeks postdenervation, but [3H]QNB binding was reduced at all periods, with the reduction from control values at 2 weeks being 34%, and at the subsequent intervals, 24-26%. The increase in number of beta adrenoceptors of the parotid gland was not related to the kind of sympathectomy (chemical or surgical) or neonatal age at which it was done; however, duration of the denervation for 2-3 weeks was necessary for the receptor increase to occur. In the adult, however, the duration of the denervation was of no importance since change in number of beta adrenoceptors did not occur at 1, 2, 3, or 4 weeks after surgical denervation but did occur after only 1 week after of reserpine-induced denervation. QNB binding was decreased with surgical sympathectomy as well as reserpine-induced sympathectomy of adult parotid gland; norepinephrine concentration was decreased to levels of a few percent of innervated glands. The relation between development of glandular supersensitivity and increase in beta adrenoceptors is discussed.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

In untreated, fasting animals the cells of the serous demilunes of the sublingual gland incorporate [3H]-leucine at a higher rate than any other of the 5 main cell types of the 3 major salivary glands. The acinar cells of the submandibular and the mucous cells of the sublingual gland show intermediate values, while the cells of the granular ducts of the submandibular and the acini of the parotid gland have a low rate of incorporation. In fasting animals extrusion of newly synthesized protein starts early in the cells of the serous demilunes. It starts between 4 and 7 hrs after [3H]-leucine injection in the acinar cells of the submandibular gland, while the other cell types did not lose substantial amounts of labelled (glyco)protein within 7 hrs. The secretion of protein is stimulated by the cholinergic drug pilocarpine in all but one of the 5 types of salivary gland cells studied. The acinar cells of the submandibular gland react strongly, the granular duct cells less strongly. Still less are the reactions of the acinar cells of the parotid and of the nucous cells of the sublingual gland. The cells of the serous demilunes of the latter appear to be insensible to pilocarpine. The effect of food uptake on secretion does not differ from pilocarpine stimulation, with one exception: the acinar cells of the parotid gland react more strongly on food uptake than on cholenergic stimulation.     

Underestimation of D-glucose phosphorylation as measured by 3H2O production from D-[2-3H]glucose

In rat pancreatic islets, tumoral islet cells (RINm5F line), parotid gland, and in human erythrocytes, but not in rat hepatocytes, the production of 3H2O from D-[2-3H]glucose is 20-30% lower than from D-[5-3H]glucose. This coincides with the production of tritiated lactic acid from D-[2-3H]glucose and may be attributable to an intramolecular hydrogen transfer in the phosphoglucoisomerase reaction. It is concluded that the production of 3H2O from D-[2-3H]glucose is not a reliable tool to assess the total rate of hexose phosphorylation.     

Differential effects of ovarian steroids and triphenylethylene compounds on macromolecular uptake and thymidine incorporation in the mouse uterus

In the rodent uterus, estrogen elicits a biphasic response i.e. an early phase (Phase I) and a late phase (Phase II). Estradiol-17 beta (E2) and estriol (E3), as well as triphenylethylene (TPE) compounds, CI-628 and clomiphene citrate (CC), were used to characterize Phase I and Phase II responses in uterine preparation for implantation in the mouse. While uterine macromolecular uptake (vascular permeability), a Phase I response, was studied in progesterone (P4)-primed animals, uterine [3H]thymidine incorporation (DNA synthesis), a Phase II response, was investigated with and without P4-priming. In the P4-primed uterus, all compounds, except CC, significantly increased uterine macromolecular uptake as determined by interstitial tissue accumulation of [125I]bovine serum albumin [( 125I]BSA). DNA synthesis as determined by cellular incorporation of [3H]thymidine was modulated by P4, estrogens and TPE compounds in a cell-type specific and temporal manner. As a single injection and in the absence of P4, E2 induced [3H]thymidine incorporation in the luminal and glandular epithelium at 18 and 24 h. E3 was inferior to E2 in this response. On the other hand, treatment with P4 for 1 day or 4 days induced [3H]thymidine incorporation primarily in stromal cells. However, stromal cell incorporation was potentiated when P4 treatment was combined with estrogens or TPE compounds. These results reveal the relative importance of Phase I and cell-type specific Phase II responses in uterine preparation for implantation.     

Intracellular cholesterol accumulation is accompanied by enhanced proliferative activity of human aortic intimal cells

Cholesterol accumulation in smooth muscle cells of unaffected human aortic intimal tissue occurred in the following conditions: (1) incubation of cells with atherogenic blood serum from patients with coronary heart disease (CHD), (2) cultivation of cells in the presence of insoluble associates containing low density lipoprotein (LDL). Preincubation of cells with blood serum from CHD patients resulted in a 2-5-fold increase in intracellular cholesterol and in 1.5-3-fold increase in cellular [3H]thymidine uptake. Blood serum collected from healthy donors had no significant effect on cultivated smooth muscle cells. When intimal cells were preincubated with insoluble associates containing LDL and components of fibrous extracellular matrix, the level of intracellular cholesterol increased from 2-4 times and uptake of [3H]thymidine increased 1.5-2.5-fold. Thus, a strong correlation was found between [3H]thymidine incorporation and intracellular cholesterol accumulation. The current study suggests that intracellular lipid accumulation may stimulate the proliferative activity of human aortic intimal cells from uninvolved tissue.     

Sympathetic denervation impairs agonist-stimulated phosphatidylinositol metabolism in rat parotid glands.

Substance P, muscarinic and alpha-adrenoceptor agonists stimulated the incorporation of [3H]inositol into phosphatidylinositol in rat parotid gland slices. Surgical denervation of the sympathetic input to the rat parotid gland by superior cervical ganglionectomy produced marked reductions in these responses. The stimulated incorporation of radiolabelled precursors into phosphatidylinositol is a measure of its resynthesis after receptor-mediated breakdown of inositol phospholipids. We therefore examined the enzymic site of the lesion induced by sympathetic denervation using parotid gland slices labelled with either [3H]inositol or [32P]phosphate and stimulated with substance P. Receptor-activated phospholipase C attack upon [3H]inositol phospholipids was assayed by measuring the formation of [3H]inositol 1-phosphate in the presence of 10 mM-Li+ to inhibit further breakdown. It was not affected by denervation. Substance P elicited a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and this response was reduced in the denervated gland. The second step in stimulated phosphatidylinositol turnover, phosphorylation of diacylglycerol to phosphatidate was not affected by denervation. Sympathetic denervation appears to induce a specific enzymic lesion in the parotid gland that impairs receptor-stimulated resynthesis of phosphatidylinositol from phosphatidate. This change in membrane lipid metabolism may be related to a number of the effects of sympathetic denervation, such as agonist supersensitivity, reduced gland cell proliferation and induction of new surface receptors.     

Autonomic receptors and cyclic nucleotides of rat parotid gland following simultaneous electrical stimulation of its parasympathetic and sympathetic nerves

[3H]dihydroalprenolol and [3H]quinuclidinylbenzilate binding of membranes of rat parotid gland were generally unchanged after 10, 15, 30, or 60 min of simultaneous electrical stimulation of the parasympathetic and sympathetic nerves to the gland, although stimulation of either nerve separately caused nerve-specific changes in both. Concentrations of cyclic nucleotides of the gland were, however, increased significantly from levels of the unstimulated parotid gland. Cyclic GMP showed a 10-fold increase after 10 min of stimulation, whereas only a 2-fold increase in cyclic AMP was found at this time. The increases were maintained, albeit at reduced levels, at 15 and 30 min also but by 60 min both were not different from levels of the unstimulated gland. The increases induced by separate stimulation of each nerve were greater but nerve specific, and the changes induced with simultaneous stimulation tended to reflect a reigning influence of one nerve on the other.     

Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.