Effects of lidocain on the membrane of human lymphocytes

(1) Lidocain inhibited [³H]thymidine and [³H]uridine incorporation in human tonsillar lymphocytes known to synthetize DNA in the absence of mitogens. This effect of lidocain was shown to be due to an inhibition by the anaesthetic of the uptake of the labeled precursors. (2) Lidocain was found to sensitize lymphocytes to the cytolytic effect of antilymphocyte serum. Lidocain is assumed to inhibit a hypothetic repair mechanism responsible for the integrity of the plasma membrane.     

Regulation of cyclic AMP level and synthesis of DNA, RNA and protein by quercetin in Ehrlich ascites tumor cells.

Quercetin (3.3, 4′, 5.7-pentahydroxy flavone) at the concentration of 10^?4 M as well as 10^?2 M theophylline and 10^?3 M dibutyryl cyclic AMP caused at least 85 per cent inhibition of [³H]thymidine incorporation in Ehrlich ascites tumor cells. At the same concentrations, these drugs decreased [³H]uridine and [³H]-L-leucine incorporation by 50–60 per cent and 35–45 per cent respectively. Ouabain (10^?3 M), the specific inhibitor of Na⁺-K⁺ pump system, did not alter the incorporation of [³H]thymidine and [³]uridine, but decreased the incorporation of [³H]-L-leucine in these cells. Treatment of Ehrlich ascites tumor cells with the polyanion dextran sulfate did not change the inhibitory effect of quercetin, theophylline and dibutyrlyl cyclic AMP on [³H]thymidine incorporation. On the other hand, this polyanion decreased the inhibitory effect of these drugs on incorporation of [³H]uridine and abolished completely their effect on incorporation of [³H]-L-leucine.     

The effect of external deoxyribonucleosides on deoxyribonucleoside triphosphate concentrations in human lymphocytes.

The effects of deoxyribonucleosides on the intracellular levels of deoxyribonucleoside triphosphates (dNTP) and on the rate of labelled thymidine incorporated into DNA of human phytohaemag-glutinin-stimulated lymphocytes have been studied. Thymidine (10^?2–10^?6M) expanded the dTTP and reduced dATP and dCTP levels. Deoxycytidine (10^?3M) expanded the dCTP level and caused inhibition of [³H] thymidine incorporation into DNA but had no detectable effect on the other dNTP concentrations. Deoxyadenosine (10^?3 M) expanded the dATP level, and reduced the other dNTP levels and deoxyguanosine (10^?4M) expanded the dGTP level and reduced the dCTP level; both inhibited [³H] thymidine incorporation into DNA. The sensitivity of these cells to the addition of deoxynucleosides to their culture medium indicates that the plasma and tissue levels of nucleosides may profoundly influence DNA synthesis by human cells in vivo.     

Cytotoxicity of antineoplastic agents on rabbit corneal epithelium in vitro

Abstract

We studied the cytotoxic effects of antineoplastic agents in vitro including cytosine arabinoside (Ara-C), methotrexate, melphalan, 5-fluorouracil (5-FU), and triethylenethiophosphoramide (thiotepa), using rabbit corneal epithelial cell primary tissue cultures. Twenty-four hour [³H]thymidine incorporation was measured 6 hr after the addition of drug or vehicle. A dose-response effect was noted for all agents. Significant (p <0.05) inhibition of thymidine incorporation was reached with Ara-C (10^?7M), methotrexate (10^?3M), melphalan (10-⁶M), 5-FU (10^?4M), and thiotepa (10^?4M). Visual inspection of the cultures was a less sensitive indicator of drug-induced cytotoxicity; except for melphalan, drug concentrations much greater than those that inhibited thymidine incorporation did not alter cell morphology. At a concentration of 10^?4M, 2′-deoxycytidine, a competitive inhibitor of Ara-C, protected corneal epithelial cells exposed to Ara-C concentrations up to 10^?5M. When used alone, concentrations of deoxycytidine greater than 10^?4M were associated with significant impairment of thymidine uptake by cells. This in vitro model may be useful in quantitatively evaluating the epithelial cytotoxicity of other current and future antineoplastic agents, as a means for predicting and possibly moderating corneal toxicity of these agents clinically.     

Acute effects of mercuric compounds on cultured mammalian cells.

The observed acute effects of methyl mercuric chloride on cultured mammalian cells were: (1) retardation of cell multiplication. (2) cell killing, (3) depression of [³H]thymidine and [³H]uridine uptake, and (4) induction of single-strand scissions of DNA. Among the cellular responses studied, [³H]thymidinc and [³H]uridine incorporation were the most sensitive indicators of cellular mercuric poisoning. Toxicities of three mercuric compounds—methyl mercuric chloride, phenyl mercuric acetate and mercuric chloride —were compared by using two indicators, [³H]thymidine incorporation and cell multiplication. Methyl mercuric chloride and phenyl mercuric acetate were equally toxic, while mercuric chloride was the least toxic. Addition of glutathione to cells pretreated with methyl mercuric chloride allowed the cells to recover from the toxic effects of methyl mercuric chloride at a faster rate than those left without glutathione.     

Effect of 4-chlorobiphenyl on substrate transport and phospholipid metabolism in mouse L5178Y lymphoma cells

The toxicity of 4-chlorobiphenyl, a constituent of Aroclor 1221, was studied in mouse L5178Y cells, in vitro. 4-Chlorobiphenyl had a varied effect on the uptake of small precursor molecules. Uptake of [³H]l-leucine, [³H]l-serine, [³H]uridine and [³H]thymidine was inhibited, while that of [³H]inositol was stimulated. There was no significant effect on either [¹⁴C]ethanolamine or [¹⁴C]choline uptake. However 4-chlorobiphenyl significantly inhibited incorporation of [¹⁴C]ethanolamine into phosphatidylethanolamine and caused a 2- to 3-fold stimulation in the incorporation of [¹⁴C]choline into phosphatidylcholine. This effect on phosphatidylcholine metabolism depended on the adsorption and continued presence of 4-chlorobiphenyl on the cell plasma membrane. The stimulation of [¹⁴C]choline incorporation was reversed when treated cells were placed in fresh growth medium under conditions where 95 per cent of the 4-chlorobiphenyl was desorbed from the cell surface. The effect of 4-chlorobiphenyl on substrate uptake and phospholipid metabolism appears to depend upon the interaction of the agent with the cell membrane surface.     

Inhibition of human lymphocyte transformation by two aryloxyalkylamidines

The incorporation of [3H]thymidine into human lymphocytes stimulated by phytohaemagglutinin (PHA) was inhibited by anilino-N-2-m-chlorophenoxypropylacetamidine (501C) and xylamidine. These amidines antagonize 5-HT, but 5-HT did not alter [3H]thymidine incorporation. 501C inhibited PHA-induced lymphocyte transformation as observed by [3H]thymidine incorporation, [3H]uridine incorporation, [3H]leucine incorporation, DNA content, potassium content, and histological examination. 501C also inhibited increased [3H]thymidine incorporation in human mixed lymphocyte cultures. The IC50 of 501C for inhibition of these processes lay between 4 and 8 microM. When added late in culture (after 6-8 h) 501C was less effective. Possible mechanisms by which 501C inhibits transformation are discussed.     

The effects of chlorambucil on the utilization of exogenous thymidine by tumour cells.

Treatment of an alkylating agent-sensitive strain of the Yoshida ascites sarcoma with chlorambucil resulted in an inhibition of the incorporation of [³H]thymidine into DNA, which could be overcome by incubating cells in high extracellular concentrations of thymidine. Increase in cellular DNA content and the dilution of specific radioactivity in pre-labelled DNA indicated that DNA synthesis was continuing at times when [³H]thymidine incorporation was inhibited. Uptake and phosphorylation of thymidine were not impaired by the treatment and the reduced incorporation of [³H]thymidine into DNA is attributable to a block in the utilization of TTP derived from exogenous nucleoside.     

Effect of slow and rapid cystometry on in vitro rat urinary bladder DNA synthesis

1. Partial outflow obstruction induces marked changes in detrusor contractile function and morphology. One common finding in all experimental animal models of partial outflow obstruction is a significant increase in bladder mass.2. Previous studies have demonstrated that partial outlet obstruction induces a rapid and substantial increase in [³H]thymidine incorporation into virtually all cellular elements of the bladder.3. The present study was designed to investigate the [³H]thymidine uptake and localization induced by exposure of the in vitro whole rat bladder model to various intravesical pressures and rates of intravesical infusion.4. The results are as follows:(a) There were no differences in DNA concentration between control and other groups.(b) Slow infusion induced a mild increase in DNA synthesis ([³Hthymidine incorporation) at 0.5 ml and a significantly greater level of DNA synthesis at 1.6 ml.(c) [³H]thymidine incorporation was significantly increased by exposure to 7.5 cm H₂O, 15 cm H₂O, and 30 cm H₂O.(d) Exposure to 60 cm H₂O and 90 cm H₂O did not initiate an increase in [³H]thymidine incorporation.(e) Autoradiography showed that all tissue elements (urothelium, connective tissue, smooth muscle) participated in the response.     

A comparison of the inhibitory effects of new non-tricyclic amine uptake inhibitors on the uptake of norepinephrine and 5-hydroxytryptamine into synaptosomes of the rat brain

The effects of new non-tricyclic amine uptake inhibitors, FS32 and FS97, on the uptake of [³H]-norepinephrine (NE) into the hypothalamic synaptosomes and [³H]-5-hydroxytryptamine (5-HT) into whole brain synaptosomes were studied. Their effects were compared with those of tricyclic antidepressants. The uptake of [³H]-NE was inhibited competitively by FS32 and FS97 with a respective K_i value of 6.5 × 10^?7 M and 3.8 × 10^?7 M. The potency of FS32 and FS97 to inhibit this uptake was almost comparable to that of clomipramine and imipramine, respectively. In the case of [³H]-5-HT uptake, FS32 and FS97 also showed competitive inhibition with a respective K_i value of 2.9 × 10^?6 M and 5.9 × 10^?6M. The ability of FS32 to inhibit [³H]-5-HT uptake was almost equal to that of nortriptyline, while FS97 was two times more potent than iprindole in inhibiting this uptake.     

The selective endothelin ETA receptor antagonist BQ123 antagonizes endothelin-1-mediated mitogenesis

The mitogenic effects of endothelin isopeptides and the selective ET_A receptor antagonist BQ123 were evaluated in rat aortic vascular smooth muscle cells. Endothelin-1 (ET-1) and endothelin-3 (ET-3) produced concentration-dependent increases in [³H]thymidine incorporation (EC₅₀ = 0.1 and 25 nM, respectively). The ET_B-selective agonist sarafotoxin 6c did not produce significant effects on [³H]thymidine incorporation. BQ123 produced selective and concentration-dependent inhibition of ET-1-mediated [³H]thymidine incorporation. These data demonstrate that ET-1-mediated mitogenesis in vascular smooth muscle is mediated by ET_A receptors.     

Comparison of some biochemical effects of teratogenic doses of mercuric mercury and cadmium in the pregnant rat

Mercuric mercury (Hg²⁺), like cadmium (Cd²⁺), interferes with the transport of certain essential metals to the conceptus in the pregnant Wistar rat and, at 48 h after the IV injection of a teratogenic dose (0.79 mg Hg²⁺/kg body weight) on day 12 of gestation, the foetal concentrations of Zn²⁺, Cu²⁺ and Fe³⁺, but not of Mg²⁺, are reduced significantly. Both Hg²⁺ and Cd²⁺, at teratogenic dose levels, inhibit the placental and foetal uptake of ⁶⁵Zn²⁺ and ⁶⁷Cu²⁺, but possibly by different mechanisms. In addition, the effects of Hg²⁺, at different times after dosing, on the uptake of these labelled tracers and of ⁵⁹Fe³⁺, administered as 15-min pulses, do not parallel the changes in the placental and foetal concentrations and contents of the endogenous, stable metallic ions. The teratogenic dose of Hg²⁺ inhibits the placental and foetal uptake of L-[4,5-³H]-leucine, but not the incorporation of the labelled amino acid into foetal protein. In contrast, the corresponding dose of Cd²⁺ inhibits both leucine uptake and protein synthesis in the placenta and foetus. Similarly, Cd²⁺ inhibits the uptake of [2-¹⁴C]-thymidine and its incorporation into foetal DNA, whereas Hg²⁺ reduces the placental and foetal uptake, but has little or no effect on the utilization of the nucleoside. Since both Cd²⁺ and Hg²⁺ reduce the foetal uptake of ⁶⁵Zn and the foetal concentration of Zn, but only Cd²⁺ interferes with DNA synthesis, it is unlikely that the inhibition of the metabolism of thymidine can be attributed to reduction in thymidine kinase activity in consequence of foetal Zn deficiency. It is concluded that the small amount of Cd²⁺ that is taken up by the foetus has a direct effect on the synthesis of DNA and protein, whereas Hg²⁺ primarily affects placental transport processes.     

Lymphoid suppression by cis-platinum(II)amines: What are the active agents?

Cisplatin and its various hydrolysis products were tested in vitro for their effects on the incorporation of [³H]thymidine into lymphocytes isolated from thymuses, spleens and stimulated lymph nodes of rats. Neither cisplatin nor the μ-hydroxo-bridged oligomers formed after hydrolysis significantly inhibited thymidine incorporation at pH7.4. However, freshly neutralised cis-diaquodiammineplatinum(II) was a potent inhibitor of thymidine incorporation by all three lymphocyte populations.In other experiments, rats were given either cisplatin or one of its hydrolysis products i.p. Cells isolated 17 hr later from the thymuses of all of these animals incorporated much less [³H]thymidine into DNA in vitro than thymocytes from saline-injected control animals. None of the platinum species significantly affected either [³H]uridine incorporation or the oxidation of [¹⁴C]octanoate to ¹⁴CO₂ by the thymocytes.Evidence for anation of di- and tri-nuclear μ-hydroxo-bridged platinum(II)amines by chloride has been obtained from spectrophotometric analyses and ¹⁹⁵Pt-NMR studies. Thiols also reacted with these platinum complexes at different rates (cis-[(NH₃)2Pt(H₂O₂)]²⁺ > derived oligomers > cisplatin).Various mechanisms for lymphoid suppression by cisplatin and its hydrolysis products are considered. It is proposed that cisplatin and its μ-hydroxo-bridged derivatives owe their lymphotoxic activity primarily to their in vivo transformation to platinum species containing aquo ligands.     

C-type natriuretic peptide inhibits rat mesangial cell proliferation by a phosphorylation-dependent mechanism

We studied the effects of C-type natriuretic peptide (CNP) on rat cultured mesangial cell proliferation. (1) Exposure to CNP (10 nM–1 μM for 72 h) inhibited [³H]thymidine incorporation into mesangial cells in a concentration-dependent manner. Atrial natriuretic peptide (1 nM–1 μM), a peptide related to CNP, also decreased [³H]thymidine incorporation into these cells in a concentration-dependent manner. (2) Both CNP (10 nM-1 μM) and atrial natriuretic peptide (10 nM-1 μM) also decreased mesangial cell number. (3) The cyclic GMP analog, 8-bromo-cyclic GMP (100 μM and 1 mM), mimicked the inhibitory effects of CNP and atrial natriuretic peptide on [³H]thymidine incorporation into mesangial cells, whereas inhibitors of protein kinase C, protein kinase A, and protein kinase G reduced the effect of both natriuretic peptides. Moreover, the phoshpatase inhibitor, calyculin A, increased [³H]thymidine incorporation into mesangial cells. (4) CNP and atrial natriuretic peptide decreased interleukin-1-, interleukin-6-, platelet derived growth factor-, angiotensin II-induced [³H]thymidine incorporation into mesangial cells. These results suggest that CNP exerts inhibitory effects on mesangial cell proliferation and that this effects depend on protein phosphorylation pathways. Received: 17 March 1997 / Accepted: 29 August 1997     

Effects of methotrexate on thymidine triphosphate levels in Chinese hamster cell cultures.

Chinese hamster ovary cells were incubated in medium containing hypoxanthine and glycine and supplied with 10^?5 M methotrexate to inhibit the endogenous synthesis of thymidine nucleotides. Within 1–5 min after addition of the inhibitor, incorporation of [6-³H]deoxyuridine into DNA was as low as 1 to 1.5 per cent of control values, indicating that endogenous synthesis of thymidine nucleotides was blocked rapidly and almost completely. The cellular dTTP content was determined under various culture conditions as a function of time after addition of methotrexate. If the medium used contained undialyzed fetal calf serum, dTTP levels decreased relatively slowly in asynchronous as well as in synchronous S-phase cell populations. Similar results were obtained with asynchronous cultures incubated in medium with dialyzed serum. In contrast, if synchronous cultures consisting predominantly of cells in S phase were incubated in medium containing dialyzed serum and supplied with methotrexate, dTTP levels decreased within 15 min from control values of 15–20 pmoles/10⁶ cells to levels of 1.5 to 3 pmoles/10⁶ cells or lower. The previously reported failure of methotrexate to cause a rapid depletion of cellular dTTP may reflect, therefore, maintenance of thymidine nucleotide pools by cells that are not in the S phase and/or uptake by cells of thymidine present in undialyzed serum used for preparation of culture media.     

Reverse trans-synaptic regulation of neuronal noradrenaline uptake.

It was established that the blocking agent of β-adrenoceptors, propranolol (5.10^?7–1.10^?6 M), activates [³H]noradrenaline uptake by isolated rat organs (vas deferens, spleen, small intestine, atrium, uterus) by 30–180 per cent. The blocking agent of α-adrenoceptors, phentolamine (5.10^?7–1.10^?6M), activates noradrenaline uptake by 30–55 per cent only in the organs possessing postsynaptic α-adrenoceptors (vas deferens, spleen, small intestine). The activator of β-adrenoceptors, isopropylnoradrenaline (5.10^?7–1.10^?6M), was shown to produce a decrease in [³H]noradrenaline uptake by 15–50 per cent in all the organs investigated. The substances activating a-adrenoceptors—urea (2.10^?3–5.10^?3M) and mesatone (1.10^?6–1.10^?5M)—inhibit [³H)noradrenaline uptake in the organs with postsynaptic α-adrenoceptors by 20–45 per cent. Activation of neuronal [³H]noradrenaline uptake induced by phentolamine (1.10^?6M) is due to the release of a humoral factor from the effector cell and its influence on adrenergic neurone. Mesatone (1.10^?5M) causes the formation and release of a humoral factor inhibiting neuronal [³H]noradrenaline uptake. The formation of humoral factors changing the intensity of amine uptake is related to the activation of protein synthesis in the effector cell. The possible mechanism of the reverse trans-synaptic regulation of neuronal noradrenaline uptake via the adrenoceptors of the effector cell is discussed.     

Synergistic effect of 5-fluorouracil and N-(phosphonacetyl)-l-aspartate on cell growth and ribonucleic acid synthesis in a human mammary carcinoma

The biological effects of $\text{N-(}\text{phosphonacetyl}\text{)-}\text{l}\text{-}\text{aspartate}$ (PALA) and 5-fluorouracil (5-FU) were examined singly, and in combination, on the growth of a human mammary carcinoma (MDA) cell line in culture. All combinations of 5-FU (2.5 × 10^?7 to 1.5 × 10^?5M) and PALA (6.0 × 10^?5 to 3.6 × 10^?3 M) resulted in synergistic inhibition of cell growth as revealed by a 50 per cent isobologram.To examine the biochemical basis for the synergism, measurements of the incorporation of [³H]-5-FU into total non-poly(A)- and poly(A)-RNA, and of the simultaneous incorporation of [¹⁴C]deoxyguanosine and [³H]deoxyuridine into DNA, were determined. The combination of 3.7 × 10^?5M PALA and 1 × 10^?6 M 5-FU produced 65–85 per cent inhibition of cell growth after continuous treatment for 13 days. Treatment of the cells for 3 or 24 hr with the same drug regimen produced approximately a 170 per cent increase in the incorporation of 1 × 10^?6M [³H]-5-FU into poly(A)RNA in comparison to [³H]-5-FU treatment alone; exposure for 24 hr to 3.7 × 10^?5 M PALA and 1 × 10^?6 M [³H]-5-FU resulted in a 285 per cent increase in the incorporation of [³H]-5-FU into non-poly(A)RNA. The incorporation of either [¹⁴C]deoxyguanosine or [³H]deoxyuridine into DNA was not inhibited by this drug regimen; however, the incorporation of [³H]deoxyuridine into DNA was elevated significantly upon 12 or 24 hr of exposure to PALA alone. PALA and 5-FU treatment resulted in a 75 per cent reduction in the concentration of UTP and no change in the concentration of 5-fluorouridine-5′triphosphate 5-FUTP) versus 5-FU treatment alone. Thus, the proportion of 5-FUTP in the total 5FUTP + UTP pool was enhanced more than 3-fold by the combination regimen. These results indicate that the synergistic effect of the combination of PALA and 5-FU on the growth of MDA cells correlates with an increased proportion of 5-FUTP in the pyrimidine nucleotide pool and, consequently, with an enhanced incorporation of 5-FU into RNA, but not with inhibition of DNA synthesis.     

The effects of antidepressants on the retention and metabolism of [3H]-norepinephrine in rat brain slices

Tricylic antidepressants acutely decrease the neuronal retention of [³H]-norepinehrine ([³H]-NE) by blocking neuronal membrane uptake and/or vesicular uptake and binding. To distinguish between effects upon the plasma membrane and upon the vesicular membrane, the retention, deamination, and O-methylation of [³H]-NE by rat brain slices were investigated in the presence of several antidepressant agents. The effects of antidepressants were compared to those of the prototype inhibitors, cocaine and reserpine, using slices of hypothalamus, brainstem. parietal cortex and caudate nucleus. Cocaine, which inhibits neuronal membrane uptake, decreased both the deamination and retention of [³H]-NE, while O-methylation was increased. Reserpine, which inhibits vesicular transport and binding, increased deamination, while it reduced retention without affecting the 0-methylation of [³H]-NE. The effects of desipramine, a prototype tricyclic antidepressant, were found to depend on the concentration. At low concentrations (10^?9-10^?8M), desipramine inhibited the retention and deamination of [³H]-NE in each brain region except the caudate. At higher concentrations (10^?7-10^w?4M), the retention of [³H]-NE was reduced further. However, deamination was increased in the caudate and, in the other three regions, deamination did not decrease further. Nortriptyline and protriptyline had actions similar to desipramine, whereas, iprindole did not affect [³H]-NE retention. These results suggest that tricyclic antidepressants are not specific selective inhibitors of neuronal membrane transport.     

Biological activities and modes of action of 9-alpha-D-arabinofuranosyladenine and 9-alpha-D-arabinofuranosyl-8-azaadenine

From earlier studies it is known that 9-α-d-arabinofuranosyladenine (α-araA) and 9-α-d-arabinofuranosyl-8-azaadenine (α-ara-8-azaA) bave in vitro antiviral activity, are cytotoxic, and are metabolized in mammalian cells to the triphosphates. This study was designed to compare the in vivo antiviral activities of these compounds and their loci of action with those of 9-β-d-arabinofura-nosyladenine (β-araA). the latter compound selectively inhibits DNA synthesis in intact cells, and its triphosphate is a known inhibitor of DNA polymerases and ribonucleotide reductase. Whereas β-araA was significantly effective in the treatment of systemic herpes simplex virus type 1 (HSV-1) infections in mice, α-araA and α-ara-8-azaA were therapeutically ineffective. α-AraATP at a concentration of ~1 mM did not inhibit (1) DNA polymerases present in crude extracts of cultured H.Ep.-2 cells; (2) DNA polymerases present in extracts of KB cells; (3) partially purified DNA polymerase-α from mouse embryo cells; or (4) DNA polymerases induced by HSV-1 and HSV-2. DNA polymerase-β from mouse embryo cells was inhibited to a small extent by 10^?4 M α-araATP. In contrast, all of these enzymes were inhibited by β-araATP at a concentration of 10^?5M (as shown in these or in earlier studies). the reductions of CDP and UDP by ribonucleotide reductase from L1210 cells were not inhibited by αaraATP (~10^?3M), whereas β-araATP produced 70–80 per cent inhibition at this concentration. In cultured H.Ep.-2 cells, α-ara-8-azaA inhibited the incorporation of thymidine, uridine, and formate into macromolecules, but it was without effect on the incorporation of adenine and hypoxanthine, and produced marginal inhibition of the incorporation of leucine. α-Ara-8-azaA produced a dose-dependent inhibition of the accumulation of [¹⁴C] formyl-glycinamide ribonucleotide in H.Ep.-2 cells treated with azaserine and [¹⁴C] formate. These results indicate that the α-nucleosides inhibit nucleic acid synthesis by mechanisms different from those of β-araA.     

Differential toxicity of carrier-bound methotrexate toward human lymphocytes,marrow and tumor cells

Methotrexate that was covalently linked to poly-l-lysine (mol. wt 3,000 and 60,000) (MTX-PLL 3K and 60K) was more inhibitory to the growth of five cell lines from human solid tumors (IC₅₀ 5?10 × 10^?8 M and 1?2.6 × 10^?8 M respectively) than to the growth of five lines of human lymphocytes (IC₅₀ 5?8 × 10^?7 M and 2?5 × 10^?7 M). In contrast, both methotrexate that was covalently linked to human serum albumin (MTX-HSA), and the free drug, were equally toxic to the two classes of cells, with IC₅₀ of 3?15 × 10^?7 M and 2?7 × 10^?8 M, respectively, for the cell types. Uptake studies showed that, whereas MTX and MTX-HSA were transported equally well into WI-L2 lymphocytes, human bone marrow cells, and an astrocytoma tumor line, uptake of MTX-PLL by the astrocytoma cells at 37° was three to four times greater than uptake by WI-L2 lymphocytes or marrow cells. [³H]Deoxyuridine ([³H]-Urd) incorporation studies indicated that low concentrations of MTX-PLL 60K (5 × 10^?7 M) resulted in inhibition of the target enzyme dihydrofolate reductase (DHFR) in the astrocytoma cells, but no iinhibition of DHFR occurred in WI-L2 lymphocytes or marrow cells until concentrations were reached where the carrier itself became toxic (5 × 10^?6 M). Two inhibitors of the lysosomal enzymes, chloroquine and lupuptin, were able to reverse the toxicity of MTX-PLL 60K against the astrocytoma cell line, increasing its IC₅₀ from 2 × 10^?8 to 2 × 10^?7 M. Both lysosomal inhibitors had no effect on the toxicity of MTX-PLL 60K against the WI-L2 lymphocytes or of MTX or MTX-HSA against either cell type, indicating that the increased toxicity of MTX-PLL 60K against the tumor cells was due, in part, to the ability of the lysosomes of these cells to convert MTX-PLL 60K either to the free drug or to a derivative that was effective in inactivating DHFR. These results suggest that comparable differential toxicity between marrow and tumor cells might also be achieved in vivo if MTX-PLL is infused over long periods at a rate that would maintain a constant serum concentration sufficient to kill tumor cells without affecting bone marrow cells.