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.     

Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2',2'-difluorodeoxycytidine

The new deoxycytidine analogue 2',2'-difluorodeoxycytidine (dFdC) is a specific inhibitor of DNA synthesis that has marked cytotoxicity and therapeutic activity. A 2-hr incubation with 0.1-10 microM dFdC decreased cellular viability 78-97%. This treatment reduced deoxynucleoside triphosphate pools, similar to the action of the ribonucleotide reductase inhibitor hydroxyurea. The most pronounced decrease occurred in the dCTP pool, quantitatively followed by the decrease of dATP, dGTP, and dTTP. In contrast, inhibition of DNA synthesis by arabinosylcytosine did not affect the dCTP level, whereas dATP, dGTP, and dTTP pools increased, but less than 2-fold. The incorporation of [5-3H]cytidine into the dCTP pool, a measure of ribonucleotide reductase activity in whole cells, was reduced to 3% of controls by 0.1 microM dFdC, but to only 40% by 0.1 microM ara-C. Each drug decreased incorporation of [5-3H]cytidine into DNA to a similar extent (greater than 94%), suggesting limitation by a reaction proximal to this step. The cellular concentration of dFdC 5'-diphosphate was 0.3 microM at 50% inhibition of the in situ activity of ribonucleotide reductase. Direct assays of partially purified ribonucleoside diphosphate reductase (EC 1.17.4.1) demonstrated 50% inhibition by 4 microM dFdC 5'-diphosphate; dFdC 5'-triphosphate was much less inhibitory. We conclude that dFdC 5'-diphosphate acts as an inhibitor of ribonucleoside diphosphate reductase.     

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     

Some effects of hydrocortisone on the early development of the rat cotton pellet granuloma.

Differential cell counts, DNA levels, cellular proliferation (studied by in vitro [³H]thymidine incorporation) and hydrolytic enzyme release into inflammatory exudates were investigated in sterile cotton wool pellets at various times after subcutaneous implantation in rats. At 2 days (the earliest time studied) the predominant cell type was the polymorphonuclear leucocyte (PMNL), but by 5 and 7 days increasing numbers of mononuclear cells (macrophages and lymphocytes) were present. Total DNA levels were almost constant, usually increasing slightly with time. DNA synthesis did not occur before day 3 and was still increasing at day 7. The two lysosomal hydrolases measured (N-acetyl-glucosaminidase) and β-glucuronidase) were present in the cell-free pellet exudate in high concentrations, suggesting that they had been released during phagocytosis. Treatment with hydrocortisone (10 mg/kg/day p.o.) during the earlier phase of the response reduced dry granuloma weight, total DNA levels and release of hydrolytic enzymes, and delayed the onset of cellular proliferation. Steroid treatment during the later proliferative phase only (days 3 to 6), reduced dry granuloma weight, total DNA level and hydrolytic enzyme release but did not affect [³H]thymidine incorporation.     

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.     

Reversal of the cytotoxicity of 3'-amino-3'-deoxythymidine by pyrimidine deoxyribonucleosides.

Mammalian cell replication is strongly inhibited by 3′-amino-3′deoxythymidine (3′-aminothymidine). This cytotoxieity can be specifically prevented or reversed by pyrimidine 2′-deoxyribonucleosides. The addition of 50 μM 2′-deoxycytidine to L1210 cells treated with 10 μM 3′ the population doubling time from about 38 hr to 17 hr. The control cells doubled every 13 hr. Another cytotoxic effect produced by 3′-aminothymidine is a dose- and time-dependent increase in cell volume. 2′-Deoxycytidine can effectively prevent and reverse this increase. 3′-Aminothymidme appears to be a potent selective inhibitor of DNA synthesis in L1210 cells. The incorporation of [³H]thymidine into DNA was inhibited by 50 per cent at 1 μM 3′-aminothymidine, a concentration which reduced L1210 replication by about 65 per cent. The rate of incorporation of [³H] adenine into DNA, another measure of DNA synthesis, was reduced similarly by 3′-aminothymidine. and 2′-deoxycytidine eliminated this inhibition as well. An effect on RNA or protein synthesis was not detected. The incorporation of [³H] uridine or [³H] adenine into RNA, or of tritiated amino acids into protein, was not reduced by 25 μM 3′-aminothymidine. These results suggest that selective disruption of DNA metabolism may account for the cytotoxicity of 3′-aminothymidine.     

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.     

Plasma membrane inhibition of macromolecular precursor transport by THC.

10^?6to 10^?4 M of delta-9-tetrahydrocannabinol (THC) or of other cannabinoids, which all have in common the C ring of olivetol, inhibit in cultured lymphocytes incorporation of [³H]thymidine. The inhibitory effect of olivetol derivatives is related to their octanol-water partition coefficient (liposolubility). Within 15 min of incubation, THC inhibits precursor pool formation and macromolecular incorporation of thymidine, uridine and leucine. THC inhibits also [¹⁴C] aminoisobutyric acid uptake into the cell, but does not alter the cellular “leakage” of this amino acid analogue. Using the isotopic dilution technique with L 1210 murine lymphoma cells and human lymphocytes, it was observed that THC decreases [³H]thymidine uptake within fifteen seconds after addition of the drug to the culture. Experiments performed at 0° indicate that THC has no action on thymidine binding to the carrier. All of these observations suggest that THC in micromolar concentration inhibits DNA synthesis through a “non-specific” alteration of membrane configuration. This effect, due to the liposolubility of the drug, could induce eonfonnational changes of membrane-bound transport systems which would inhibit their function.     

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.     

Alteration of DNA by 5-(3-methyl-1- triazeno)imidazole-4-carboxamide (NSC-407347)

5-(3-Methyl-1-triazeno)imidazole-4-carboxamide (NSC-407347, MTIC), an active intermediate in the metabolism of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (NSC-45388, DTIC), was investigated for its effect on DNA. When [³H]MTIC was added to an aqueous solution of calf thymus DNA, it was rapidly hydrolyzed, and less than 0.5 per cent of the added ³H was recovered in DNA as methyl groups attached to its base and phosphate constituents. It was estimated that the hydrolysis of phosphotriesters in methylated DNA released 0.5 per cent of total bound ³H of DNA in the experiment in vitro, and 5.7 per cent in vivo. When MTIC was added to tissue culture cells which had been prelabeled with [³H]thymidine, there was a large and immediate increase in acid-soluble radioactivity of both the cell media and the cells. At the same time, there was an accelerated drop in the specific activity of DNA, indicating that degeneration of DNA had occurred; MTIC at 10^?3 M had a greater effect than at 10^?5 M. Paper chromatographic studies showed that the acid-soluble radioactivity was predominantly in the form of thymidine. MTIC at 10^?4 M induced repair synthesis of DNA but inhibited semi-conservative replication. The extent of repair synthesis was greater at 30 min than after 5 min. Treatment of cells with MTIC at 10^?3 M irreversibly reduced the sedimentation of DNA in alkaline sucrose gradient. At 10^?5 M, the reduction was reversible, and a normal pattern was restored in 30 min. Comparison of these results with those of sedimentation analysis of DNA in formamide gradient at neutral pH indicated that single strand breaks which were apparent in alkaline sucrose were probably the result of exposure of alkali-labile lesions in DNA to high pH.     

Deoxyribonucleoside triphosphate pools in human diploid fibroblasts and their modulation by hydroxyurea and deoxynucleosides

Deoxyribonucleoside triphosphate (dNTP) pool levels were examined in synchronized and unsynchronized log phase cultures and in quiescent cultures of human diploid foreskin fibroblasts. dNTP levels were in good agreement with those previously published for human HeLa and lymphoblastic leukemia cells. dCTP and dGTP levels showed only a modest lowering in quiescent as compared to log-phase cells, but dATP and dTTP levels were reduced dramatically in quiescent cultures. Cells synchronized by serum starvation and assayed at the peak DNA synthetic phase (18-21 hr post release) showed substantially higher pools of all four dNTPs. Hydroxyurea treatment reduced only purine dNTPs in both log phase and confluent cells while increasing dTTP and dCTP pools. The effects of deoxynucleosides on dNTP pools were also examined and are discussed in light of current models regarding regulation of purified ribonucleotide reductase formulated from in vitro studies.     

Correlation of thymidine-enhanced incorporation of ara-C in deoxyribonucleic acid with increased cell kill

The treatment of L1210 cells with 10^?3 or 10^?3 M thymidine prior to 1-β-d-arabinofurano-sylcytosine (ara-C) exposure increased significantly the incorporation of ara-C in DNA. Furthermore, the increased formation of (ara-C)DNA correlated with an enhanced loss of clonogenic survival (P < 0.0001). Thymidine pretreatment at either concentration for 16 hr was more effective than a 2-hr pretreatment. the results suggest that the increased formation of (ara-C)DNA is, in part, related to the enhancement of cells traversing through S-phase following thymidine exposure. These results extend our previous observations showing a highiy significant relationship between the extent of ara-C incorporation in DNA and loss of clonogenic survival. the incorporation of ara-C in DNA is a useful and easily measurable parameter for evaluating the effects of agents that modulate ara-C metabolism.     

Effects of 8-azaadenosine and formycin on cell lethality and the synthesis and methylation of nucleic acids in human colon carcinoma cells in culture

The cytocidal and biochemical effects of formycin and 8-azaadenosine in the presence and absence of the adenosine deaminase inhibitor, 2′-deoxycoformycin, were studied in human colon carcinoma (HT-29) cells in culture. Logarithmically growing cells were unaffected by 24-hr exposure to either 10^?6M formycin or 8-azaadenosine, but 1 to 1.4 log reductions in colony formation were produced by 10^?5M of each analog. In the presence of 10^?6M 2′-deoxycoformycin, a 3- and 30-fold potentiation of the cytocidal activity of 8-azaadenosine and formycin, respectively, was produced. Inhibition of DNA synthesis but not RNA synthesis by 8-azaadenosine paralleled its cytocidal activity; however, neither variable correlated closely with the cytotoxic effects of formycin. In addition, the methylation of nuclear RNA was unaffected by both drugs while the methylation of 5-methyl-deoxycytidine in DNA was inhibited to a lesser extent than DNA synthesis. Measurements of the incorporation of [³H]formycin and [³H]8-azaadenosine into nuclear RNA and DNA in the presence and absence of 2′-deoxycorformycin indicated that formycin substitution in RNA and DNA was enhanced 10- and 20-fold, respectively, while [³H]8-azaadenosine incorporation into both nucleic acids was increased 6- to 7-fold. These results suggest that the incorporation of formycin into nucleic acids, particularly DNA, correlates closely with its lethal effect on cell viability. On the other hand, the cytocidal activity of 8-azaadenosine more clearly parallels its inhibitory effect on DNA synthesis rather than its substitution into nucleic acids.     

Selective inhibition of herpesvirus DAN synthesis by foscarnet

The inhibition of cellular and herpesvirus DNA synthesis by phosphonoformate (INN; foscarnet sodium) has been determined after isopycnic separation of cellular and viral DNA in CsCl gradients. The DNA synthesis was determined as the incorporation of ortho[³²P]phosphate and [³H]thymidine into DNA. A 50% inhibition of herpes simplex virus DNA synthesis was observed at 50 μM phosphonoformate. At this concentration cellular DNA synthesis was not inhibited. At 500 μM phosphonoformate more than 95% of the viral DNA synthesis was inhibited, while the cellular DNA synthesis in infected and uninfected cells were inhibited to about 10%. The same results were obtained in both Vero and GMK cells and using either ortho[³²P]phosphate or [³H]thymidine to label the newly synthesized DNA. The 50% inhibitory concentration of phosphonoformate was similar for inhibition of herpes DNA synthesis and plaque reduction.     

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.     

9-Deazaadenosine—A new potent antitumor agent

9-Deazaadenosine (9-DAA), a novel purine analog, was found to be a potent inhibitor of the growth of nine different human solid tumor cell lines in vitro and of pancreatic carcinoma (DAN) in antithymocyte serum (ATS)-immunosuppressed mice. In culture, ic₅₀ values ranged from 1.1 to 8.5 × 10^?8 M. Ovarian carcinoma (MR) was the only cell line in which the activity of 9-DAA was potentiated (about 10-fold) by pretreatment with the adenosine deaminase inhibitor 2'-deoxycoformycin (dCF). After incubation of cultured pancreatic DAN cells with 9-DAA (10-^?5M) for 2 hr, a peak appeared in the triphosphate region of HPLC nucleotide profiles that was identified tentatively as 9-deazaATP. Under the same incubation conditions, the incorporation of [³H]uridine into RNA and of [³H]thymidine into DNA was inhibited by 34 and 80% respectively. In vivo studies using ATS-immunosuppressed mice showed that 9-DAA at 0.4 mg/kg/day for 3 consecutive days reduced pancreatic carcinoma (DAN) tumor weights to approximately 50% of untreated controls. The nucleoside transport inhibitor p-nitrobenzyl-6-thioinosine (NBMPR) was shown to selectively protect host tissues from 9-DAA toxicity and, thereby, potentiated the antitumor activity of 9-DAA in vivo at optimal dosages.     

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.     

Effects of chlorpromazine and actinomycin D on uptake and incorporation of certain amino acids,hypoxanthine and thymidine in cultures of human skin epithelial cells

Chlorpromazine (CPZ) 1· × 10^?4 M inhibited the uptake and incorporation of alanine (25 and 3 per cent of controls respectively), the uptake of α-aminoisobutyric acid (AIB, 39 per cent of controls) and the uptake and incorporation of hypoxanthine (36 and 44 per cent of controls) into acid-soluble and insoluble fractions of human skin epithelial cells (HE cells, NCTC 2544) grown in culture. The uptake of phenylalanine and 1-aminocyclopentane-1-carboxylic acid (cycloleucine) was not inhibited by CPZ in the same dose range, but CPZ above 10^?5 M inhibited the incorporation of phenylalanine into acid-insoluble material with 50 per cent inhibition at 6·5 × 10^?5 M. Actinomycin D stimulated the uptake of thymidine into the acid-soluble fraction of the HE-cells, 5·0 μg/ml increased the uptake to 160 per cent of the controls. The uptake of hypoxanthine was inhibited by actinomycin D, 5·0 μg/ml reduced the uptake to 67 per cent of controls. Actinomycin D did not alter the uptake of AIB or cycloleucine.     

Synergistic and antagonistic interactions of methotrexate and 1-β-D-arabinofuranosylcytosine in hepatoma cells: The modulating effect of purines

A 4-hr pretreatment with methotrexate antagonized the cytotoxic effect of subsequent arabinosylcytosine treatment in rat hepatoma cells of lines N₁S₁ and 3924A, but in the hepatoma line 8999R and the fibroblast line BF5, MTX pretreatment was synergistic with the arabinosylcytosine treatment. Measurement of cellular deoxyribonucleoside triphosphate concentrations showed that in those lines in which antagonism was found the dCTP increased, whereas in the lines where the drugs were synergistic the dCTP pool was decreased. Conversely, dATP levels were high when the drugs were synergistic and low when antagonism was obtained. Although methotrexate pretreatment antagonized arabinosylcytosine in N₁S₁ and 3924A cells, pretreatment of these cells with the combination of methotrexate plus a purine (either hypoxanthine or 2′-deoxyadenosine) resulted in synergism with arabinosylcytosine. Deoxynucleotide pool measurements showed that methotrexate in combination with either hypoxanthine or 2′-deoxyadenosine increased dATP and decreased dCTP in the N₁S₁ and 3924A hepatoma cells. In N₁S₁ cells, pretreatment with 2′-deoxyadenosine alone for 4 hr was synergistic with arabinosylcytosine. It was concluded that elevated dATP pools enhanced arabinosylcytosine cytotoxicity by depleting the dCTP pool, through feedback inhibition of ribonucleotide reductase, thus causing greater inhibition of DNA biosynthesis and greater incorporation of AraCTP into nucleic acid. Methotrexate was synergistic in those cell lines where dATP accumulated and dCTP was decreased, but when methotrexate had a potent antipurine effect dCTP pools increased and arabinosylcytosine was antagonized. The synergistic interaction was more marked at cytocidal drug concentrations than it was at growth-inhibitory doses.     

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.