Increased Cytotoxicity and Decreased In Vivo Toxicity of FdUMP[10] Relative to 5-FU

Abstract

The efficacy of treatment with 5-Fluorouracil (5-FU) is limited, in part, by its inefficient conversion to 5-Fluoro-2′-deoxyuridine-5-O-monophosphate (FdUMP). We present data indicating that FdUMP[10], designed as a pro-drug for intracellular release of FdUMP, is cytotoxic as a consequence of uptake of the multimeric form. FdUMP[10] is stable in cell culture medium, with more than one-half of the material persisting as multimers of at least six nucleotides after a 48 h incubation at 37°C. FdUMP[10] is more than 400 times more cytotoxic than 5-FU towards human colorectal tumor cells (H630). FdUMP[10] also has decreased toxicity in vivo, with doses as high as 200 mg/kg/day (qdx3) administered to Balb/c mice without morbidity, compared to a maximum tolerated dose of 45 mg/kg/day for 5-FU using the same protocol. FdUMP[10] shows reduced sensitivity to OPRTase-and TK-mediated drug resistance, relative to 5-FU and FdU, respectively, and is much more cytotoxic than 5-FU towards cells that overexpress thymidylate synthase. Thus, FdUMP[10] is less susceptible to resistance mechanisms that limit the clinical utility of 5-FU. The increased cytotoxicity, decreased toxicity in vivo, and reduced sensitivity to drug resistance of FdUMP[10], relative to 5-FU, indicates multimeric FdUMP is potentially valuable as an antineoplastic agent, either as a single agent, or in combination with 5-FU.     

Increased cytotoxicity and decreased in vivo toxicity of FdUMP[10] relative to 5-FU.

The efficacy of treatment with 5-Fluorouracil (5-FU) is limited, in part, by its inefficient conversion to 5-Fluoro-2'-deoxyuridine-5'-O-monophosphate (FdUMP). We present data indicating that FdUMP[10], designed as a pro-drug for intracellular release of FdUMP, is cytotoxic as a consequence of uptake of the multimeric form. FdUMP[10] is stable in cell culture medium, with more than one-half of the material persisting as multimers of at least six nucleotides after a 48 h incubation at 37 degrees C. FdUMP[10] is more than 400 times more cytotoxic than 5-FU towards human colorectal tumor cells (H630). FdUMP[10] also has decreased toxicity in vivo, with doses as high as 200 mg/kg/day (qdx3) administered to Balb/c mice without morbidity, compared to a maximum tolerated dose of 45 mg/kg/day for 5-FU using the same protocol. FdUMP[10] shows reduced sensitivity to OPRTase- and TK-mediated drug resistance, relative to 5-FU and FdU, respectively, and is much more cytotoxic than 5-FU towards cells that overexpress thymidylate synthase. Thus, FdUMP[10] is less susceptible to resistance mechanisms that limit the clinical utility of 5-FU. The increased cytotoxicity, decreased toxicity in vivo, and reduced sensitivity to drug resistance of FdUMP[10], relative to 5-FU, indicates multimeric FdUMP is potentially valuable as an anti-neoplastic agent, either as a single agent, or in combination with 5-FU.     

Cytotoxicity and In Vivo Tolerance of FdUMP[10]: A Novel Pro-Drug of the TS Inhibitory Nucleotide FdUMP

Abstract

The cytotoxicity of the 10mer ODN FdUMP[10] towards human colorectal tumor cells was evaluated using a clonogenic assay. FdUMP[10] was more than 100-fold more active than 5-FU at inhibiting colony formation of H630 cells. FdUMP[10] was also evaluated for cytotoxicity in the NCI 60 cell line screen, and showed markedly improved activity relative to 5-FU against numerous tumor cell lines. The in-vivo tolerance of FdUMP[10] is more than three-fold greater per mole fluorinated pyrimidine, than 5-FU.     

Positive interaction between 5-FU and FdUMP[10] in the inhibition of human colorectal tumor cell proliferation.

Interaction between 5-fluorouracil (5-FU) and FdUMP[10], a novel pro-drug formulation of the thymidylate synthase (TS) inhibitory nucleotide 5-fluoro-2'-deoxyuridine-5'-O-monophosphate (FdUMP), was investigated to evaluate the feasibility of using these two forms of fluorinated pyrimidine in combination chemotherapy regimens. 5-FU and FdUMP[10] are expected to differ in their relative intracellular distribution of active metabolites, and their combined administration may result in either a positive or a negative interactive effect. The dose-response behaviors of 5-FU and FdUMP[10] toward H630 and H630-10 (human colorectal tumor) cells were first investigated separately. Effects on cell viability were measured using an assay for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), while cytotoxicity and apoptosis were investigated using clonogenic and TUNEL assays, respectively. Exposure of H630 cells to concentrations of FdUMP[10] insufficient to inhibit cell proliferation as a single agent markedly increased the cytotoxicity of 5-FU. The results indicate that 5-FU and FdUMP[10] interact in a positive manner, and that combining these two forms of fluorinated pyrimidine may be clinically beneficial.     

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

Summary Whole-body autoradiography demonstrated the different distribution of [¹²⁵I]-C-ANP and [¹²⁵I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [¹²⁵I]-ANP and [¹²⁵I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [¹²⁵I]-ANP administration were detected, no or just few radioactivity was found after administration of [¹²⁵I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [¹²⁵I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.This work is part of the doctoral thesis of Frank Heidemann to be presented at the Ludwig-Maximilians-Universität München, FRG     

Enhanced 5-lipoxygenase activity in lung macrophages compared to monocytes from normal subjects

We compared lipoxygenase activities of lung macrophages obtained from bronchoalveolar lavage to activities of blood monocytes purified by using discontinuous plasma/Percoll density gradients and adherence to tissue culture plastic in five normal subjects. Cells were incubated with ionophore A23187 (10(-9) to 10(-5) M) or arachidonic acid (0.12 to 80 microM) for 1 to 60 min at 37 degrees C to construct dose-response and time-dependence curves of lipoxygenase product generation. Products were identified and were quantified by using high-pressure liquid chromatography and ultraviolet spectroscopy. Under all conditions of product generation, both macrophages and monocytes generated predominantly (5S,12R)-dihydroxy-(6Z, 8E, 10E, 14Z)-eicosatetraenoic acid (leukotriene B4 (LTB4] and (5S)-hydroxy-(6E, 8Z, 11Z, 14Z) - eicosatetraenoic acid (5 - HETE), but, in each subject, macrophages invariably released greater amounts of LTB4 and 5-HETE than monocytes. In response to A23187, macrophages released a maximum of 183 +/- 96 pmol of LTB4 and 168 +/- 108 pmol of 5-HETE per 10(6) cells (mean +/- SEM), whereas monocytes released only 16 +/- 1 and 18 +/- 8 pmol per 10(6) cells of LTB4 and 5-HETE, respectively. After adding arachidonic acid, macrophages released a maximum of 52 +/- 21 pmol of LTB4 and 223 +/- 66 pmol of 5-HETE, whereas monocytes released no detectable products. The results suggest that mononuclear phagocyte maturation in the lung may be accompanied by an enhanced ability to generate 5-lipoxygenase products.     

Autoradiographic localization of [125I]-C-ANP compared to [125I]-ANP in rat tissue.

Whole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.     

Investigation of the Ternary Complex Formed Between Recombinant Rat Hepatoma Thymidylate Synthase,FdUMP op S4FdUMP and N5, N10-Methylenetetrahydrofolate with the Use of 1H AND 19F NMR

Abstract

Interactions of mammalian (rat hepatoma) thymidylate synthase with FdUMP and its 4-thio analogue were studied in solution with the use of ¹H and ¹⁹F NMR. The results pointed to formation of ternary covalent complexes of TS with either nucleotide in the presence of N⁵, N¹⁰-methylenetetrahydrofolate and only noncovalent binary complexes in the absence of the cofactor.     

Biochemical and biological properties of 5-bromotubercidin: differential effects on cellular DNA-directed and viral RNA-directed RNA synthesis

We have studied the biochemical and biological properties of 5-bromotubercidin (4-amino-5-bromo-7-beta-d-ribofuranosyl-pyrrolo [2,3-d]pyrimidine) (BrTu), a synthetic analogue of the highly cytotoxic pyrrolo[2,3-d]pyrimidine ribonucleoside antibiotic tubercidin (Tu) that interferes with numerous cellular processes, and has been shown to possess biological specificity and selectivity. Thus, BrTu entered the mammalian cell nucleotide pool by phosphorylation, was incorporated into RNA in an unmodified form and, as a consequence, reversibly inhibited (15 microM) mammalian cell growth and the synthesis of high-molecular-weight cellular RNA species (i.e., mRNA and rRNA). However, BrTu (300 microM) did not inhibit picornavirus RNA synthesis or multiplication, and thus discriminated between virus RNA-dependent and all forms of DNA-dependent RNA synthesis whether of cellular or viral origin; because of this BrTu should prove valuable as a metabolic probe for studying the cell-virus relationship. Furthermore, BrTu is a substrate for adenosine kinase (K(m)=24 microM), and is also its potent inhibitor (K(i)=0.93 microM); thus, low concentrations of BrTu (1.5 microM), which did not inhibit cell growth, blocked phosphorylation and the cellular uptake of other, highly cytotoxic pyrrolo-pyrimidine nucleoside analogues (e.g., tubercidin). This block in cellular uptake and incorporation of toxic analogues was associated with the protective effect of BrTu against cell killing by the analogues, providing a mechanism by which BrTu and these analogues can, as we reported elsewhere [J. Virol.1999, 73, 6444], be used for the selective inactivation of replicating picornaviruses.     

Bacteriophage M13 DNA-directed in vitro synthesis of gene 5 protein

A method is presented which allows one to follow in a relative rapid and accurate way the synthesis of gene 5 protein of the bacteriophage M13 in a DNA-dependent cell-free system. Due to its unique property of binding selectively to single-stranded but not to double-stranded DNA, gene 5 protein can readily be separated, by means of sucrose densitygradient centrifugation, from the other polypeptides synthesized in the cell-free system. The meaning of this technique for the elucidation of the mechanism(s) which regulates gene 5 protein synthesis is discussed.     

Radiotoxicities of [3H]thymidine and of [3H]arginine compared in mouse embryos in vitro

Tritium that is bound to organic molecules is of special risk for living systems, in particular when such molecules are components of the cell nucleus. Therefore, [3H]thymidine and [3H]arginine were studied for radiotoxicity in early mammalian embryo development. Starting with the two-cell stage, mouse embryos were incubated in vitro with [3H]thymidine or [3H]arginine at either 370 Bq/ml (10 nCi/ml) or 925 Bq/ml (25 nCi/ml). Development in vitro was followed up to the formation of the inner cell mass at 192 h postconception (p.c.). There was no difference in radiotoxicity of the two substances with respect to cell proliferation; however, formation of blastocysts, hatching of blastocysts, trophoblast outgrowth, and formation of inner cell mass were impaired more strongly by [3H]arginine than by [3H]thymidine when the external exposure concentrations were the same. Similarly, micronuclei were seen in blastocysts at 96 h p.c. at higher frequency after incubation with [3H]arginine. However, uptake of [3H]arginine by the embryos was considerably faster than that of [3H]thymidine, and this most probably accounts for the apparent difference in radiotoxicity.     

Binding of [99mTc]chondroitin sulfate to scavenger receptors on human chondrocytes as compared to binding of oxidized [125I]LDL on human macrophages

Chondroitin sulfate (CS) used for treatment of osteoarthritis exerts distinct effects on human articular chondrocytes in vitro. We performed a binding analysis with 99mTc-labeled CS (Condrosulf, a commercial CS preparation containing calcium stearate) and cultured human chondrocytes in order to evaluate the presence of specific receptors. Saturation binding at 37 degrees C for 2 h revealed the presence of high-affinity binding sites for CS with a Kd of 2.3 x 10(-9) mol/L and a Bmax of 5.0 x 10(8). Extensive dialysis of Chondrosulf led to a decrease of the binding affinity by 52.5 +/- 19.5% and of the number of CS binding sites/cell by 62.0 +/- 14.0%, demonstrating that the additive present in the Condrosulf preparation enhances CS binding. The nature of the binding site is not yet known but evidence exists in the literature that the scavenger receptor CD36, thoroughly investigated on macrophages, is also found on chondrocytes and might be involved in CS binding. Therefore, we undertook a comparative binding study with human monocytes and labelled LDL and oxidized LDL, the latter being a postulated atherogenic agent in atherosclerosis. For [125I]-LDL binding we found a Kd of 0.45 x 10(-8) mol/L and a Bmax of 0.14 x 10(6) on quiescent monocytes and for [125I]-(ox)LDL binding a Kd of 1.8 x 10(-8) mol/L and a Bmax of 1.3 x 10(6) using LPS-activated monocytes. These data are comparable to the binding affinity found for lipoprotein-proteoglycan-complexes and hence are an indication but not a proof that CD36 is involved in CS binding to human chondrocytes.     

[3H]Acetylcholine and [3H]5-hydroxytryptamine release from rat midbrain slices and the effects of calcium and phenobarbital

The K-stimulated release of [³H]ACh from rat midbrain slices prelabeled by incubation with [³H]choline was dependent on extracellular Ca. Phenobarbital inhibited the K-stimulated [³H]ACh release and the IC₅₀ was equal to that found for K-stimulated endogenous ACh release. These results support the suggestion that barbiturates primarily inhibit the Ca-dependent stimulated release of ACh and affect ACh synthesis only indirectly. K-Stimulated release of [³H]5-HT was also inhibited by removing Ca from the medium or by adding phenobarbital which further supports the effects of barbiturates on the depolarization-induced release process. Fluoxetine, an inhibitor of 5-HT uptake, increased the amount of [³H]5-HT found in the medium but did not fully block the uptake of [³H]5-HT in this slice preparation.     

Differential effects of growth factors on [3H]thymidine incorporation and [125I]iodine uptake in FRTL-5 rat thyroid cells

We studied the effect of several growth factors on DNA synthesis and function of FRTL-5 rat thyroid cells by simultaneous measurement of [3H]thymidine incorporation and [125I]iodide uptake. Endothelial cell growth factor, fibroblast growth factor, platelet-derived growth factor, and insulin-like growth factor I stimulated thymidine incorporation in a dose-dependent manner without the parallel increase of [125I]iodide uptake. These growth factors had an additive effect with thyroid-stimulating hormone (TSH) on thymidine incorporation, but they inhibited TSH-stimulated iodide uptake. Bombesin stimulated thymidine incorporation and inhibited TSH-stimulated iodide uptake; epidermal growth factor and gastrin-releasing peptide 10 had neither effect. None of the growth factors studied affected iodide uptake in the absence of TSH. Of the growth factors tested, endothelial cell growth factor, fibroblast growth factor, insulin-like growth factor bombesin, and platelet-derived growth factor all share similar differential effects on FRTL-5 cells: stimulation of DNA synthesis, potentiation of the effects of TSH on DNA synthesis, and attenuation of the effects of TSH on cell function. The data suggest that these growth factors may play important roles in regulation of thyroid function.     

Radioimmunoassay for immunoreactive [des-Leu10]-angiotensin I

A specific radioimmunoassay for the angiotensin-derived peptide [des-Leu10]-angiotensin I (AI-dL) is described. Antisera obtained from rabbits injected with immunogen prepared by coupling bovine beta-thyroglobulin to the peptide with carbodiimide were specific to this peptide and did not recognise related angiotensin peptides such as AI, AII, AIII, nor did they recognise other peptides such as bradykinin, substance P, bombesin or dynorphin(1-8). Immunoreactive AI-dL was detected for the first time in the plasma of rats and humans following purification by HPLC at concentrations of 78 and 40 pg/ml, respectively. Concentrations of AI-dL are increased following chronic administration of captopril to rats.     

Genotoxic effects of the chlorinated hydroxyfuranones 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone and 3,4-dichloro-5-hydroxy-2[5H]-furanone in Tradescantia micronucleus assays

This is the first report of clastogenic effects of chlorinated hydroxyfuranones (CHFs) in plants. Two byproducts of water chlorination, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) and 3,4-dichloro 5-hydroxy-2[5H]-furanone (MA) induced a dose dependent increase of micronuclei (MN) in pollen mother cells of Tradescantia when doses up to 100 μg MX and 500 μg MA were applied directly to the inflorescences. In contrast, exposure of the stems in aqueous solutions containing up to 1 mg/I MX and 10 mg/I MA did not cause a positive response.