Robert I. Glazer

Studies on the mechanism of action of tiazofurin metabolism to an analog of NAD with potent IMP dehydrogenase-inhibitory activity.

Following the parenteral administration of tiazofurin, 2-beta D-ribofuranosylthiazole-4-carboxamide (thiazole nucleoside, TR), a potent but reversible inhibitor of IMP dehydrogenase is generated in subcutaneous nodules of the P388 leukemia. The compound responsible for this effect has been isolated from homogenates of the tumor by ion-exchange HPLC, and its presence monitored by enzyme-inhibition assay. The inhibitor has also been prepared by incubation of tiazofurin with P388 cells in culture. Chromatographically, the inhibitory principle exhibits a moderately strong set negative charge at pH 3, and elutes in the general vicinity of the nucleoside-5-diphosphates; its absorption maximum in aqueous solution (pH 7) lies at 252 nm. Exposure of the molecule to snake-venom phosphodiesterase or to nucleotide pyrophosphatase destroys its inhibitory potency, whereas other phosphodiesterases are either less effective or inert. Since these results suggested that the anabolite might be a dinucleotide with a phosphodiester linkage of the kind found in NAD, attempts were made to synthesize such an analogue from the 5-monophosphate of thiazole nucleoside and ATP-Mg2+, using a purified preparation of NAD pyrophosphorylase; modest yields were obtained of a compound with chromatographic, spectral and enzyme-inhibitory properties identical to those of the material isolated from P388 tumor nodules. This enzyme-synthesized material was radioactive when [3H]ATP was used as cosubstrate, and yielded both AMP and thiazole nucleoside-5-monophosphate on treatment with phosphodiesterase. It resisted attack by NAD glycohydrolase. An apparently identical dinucleotide was also synthesized chemically by means of the Khorana condensation. Mass spectral analysis and nuclear magnetic resonance studies with homogeneous preparations of both the enzymically and chemically synthesized compound were compatible with its being a dinucleotide in which the nicotinamide of NAD has been replaced by thiazole-4-carboxamide. Versus IMP dehydrogenase, the dinucleotide exhibited a K1 of approximately 2 X 10(-7) M and was non-competitive with NAD as the variable substrate. Other NAD utilizing enzymes, including representative dehydrogenases and poly ADP ribose polymerase, were, by comparison to mammalian IMPD, resistant to inhibition by TAD. The properties of this novel dinucleotide are compared and contrasted with those of analogs of NAD containing modifications in the pyridine, adenine or ribofuranose rings, as well as in the pyrophosphate bridge.

Studies on the mechanism of action of 2-β-d-ribofuranosylthiazole-4-carboxamide (NSC 286193)-II. Relationship between dose level and biochemical effects in P388 leukemia in vivo

Administration of the novel thiazole C-nucleoside, 2-beta-D-ribofuranosylthiazole-4-carboxamide (NSC 286193), to BDF1 mice bearing subcutaneous implants of P388 leukemia provoked a sharp depression in the concentration of intratumoral guanine nucleotides and a correspondingly large expansion of the IMP pools. Measurements of IMP dehydrogenase in the tumors of treated mice revealed that this enzyme was inhibited in a dose-responsive way, with approximately 50% inhibition engendered by the administration of the drug at a dose of 25 mg/kg and greater than 90% inhibition by all doses greater than 100 mg/kg. The inhibition of enzyme activity, seen after a dose of 250 mg/kg, reached a maximum 120 min after treatment and had subsided substantially 8 hr after dosing; by 24 hr. enzyme activity was fully restored. These results, coupled with the observation that the antitumor activity of the drug could be prevented in large part by the simultaneous administration of guanosine, support the conclusion that 2-beta-D-ribofuranosylthiazole-4-carboxamide, after anabolism, exerts its antineoplastic effects via a state of guanine nucleotide depletion. In extracts of the tumors of mice given parenteral injections of the thiazole nucleoside, a potent dialyzable inhibitor of IMP dehydrogenase was demonstrable: its concentration fluctuated in parallel with enzyme inhibition. Although the chemical identity of the proximate inhibitory species has yet to be established, it is concluded on kinetic grounds that it is neither the native nucleoside nor its 5-monophosphate.

Cyclopentenyl cytidine analogue. An inhibitor of cytidine triphosphate synthesis in human colon carcinoma cells.

The mechanism of action of the cyclopentenyl analogue of cytidine, cCyd, was investigated in human colon carcinoma cell line HT-29. Upon exposure of cells to 10(-6)M cCyd, cell viability was reduced to 20% of control, whereas cytocidal activity was not present after 2 hr of drug exposure. Cell lethality was partially reversible by Urd, Cyd or dCyd at 10(-6)M cCyd, and fully reversible by these nucleosides at 2.5 X 10(-7)M cCyd. The incorporation of [14C]dThd and [3H]Urd into DNA and RNA was inhibited by 50% by exposure for 2 hr to 2.5 X 10(-7) and 1.5 X 10(-6)M cCyd respectively. Upon 24 hr of drug exposure, the IC50 for RNA synthesis was reduced 2.5-fold, whereas DNA synthesis was almost totally inhibited. cCyd produced a rapid and preferential reduction of CTP synthesis with a half-life of 1 hr at 10(-6)M drug. The IC50 of cCyd for reducing CTP concentrations after 2 hr of drug exposure was 4 X 10(-7)M. Concomitant with the reduction of CTP levels was the inhibition of transcription of rRNA and, to a lesser extent, tRNA, without changes in the processing nucleolar RNA. No changes in the size of DNA were produced following treatment with cCyd. These results indicate that cCyd is a potent and rapid inhibitor of CTP synthesis and that this effect correlates with its cytocidal activity.

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

Abstract The biological effects of N-( phosphonacetyl )- l - 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 −5 M) 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 [ 3 H]-5-FU into total non-poly(A)- and poly(A)-RNA, and of the simultaneous incorporation of [ 14 C]deoxyguanosine and [ 3 H]deoxyuridine into DNA, were determined. The combination of 3.7 × 10 −5 M 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 −6 M [ 3 H]-5-FU into poly(A)RNA in comparison to [ 3 H]-5-FU treatment alone; exposure for 24 hr to 3.7 × 10 −5 M PALA and 1 × 10 −6 M [ 3 H]-5-FU resulted in a 285 per cent increase in the incorporation of [ 3 H]-5-FU into non-poly(A)RNA. The incorporation of either [ 14 C]deoxyguanosine or [ 3 H]deoxyuridine into DNA was not inhibited by this drug regimen; however, the incorporation of [ 3 H]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.

Measurement of S-adenosyl-l-methionine levels by SP sephadex chromatography

Cation-exchange chromatography with sulfopropyl Sephadex was used to measure the levels of S-[methyl-3H]adenosyl-L-methionine synthesized by L1210 cells in vitro. Separation of S-[methyl-3H]adenosyl-L-methionine from [methyl-3H]methionine and other metabolites was achieved by stepwise elution with varying concentrations of HCl.

Analysis of tyrosine kinase activity in cell extracts using nondenaturing polyacrylamide gel electrophoresis

A general procedure for detecting tyrosine kinase activity in crude or purified preparations using nondenaturing gel electrophoresis is presented. Samples are resolved by electrophoresis in minigels which are then incubated in an assay mixture containing [gamma-32P]ATP, poly(glutamic acid, tyrosine)4:1, and cofactors. Subsequently, gels are fixed and washed in trichloroacetic acid-pyrophosphate, dried, and analyzed by autoradiography or liquid scintillation counting. The procedure is simple and fast and allows analysis of different molecular weight forms of tyrosine kinase under linear kinetics at 37 degrees C without interference from phosphatases and proteases.

Comparison of DNA scission and cytotoxicity produced by Adriamycin and 5-iminodaunorubicin in human colon carcinoma cells.

The quinone-modified anthracycline, 5-iminodaunorubicin, which does not spontaneously generate free radicals, was compared to Adriamycin on the basis of DNA-protein crosslink-associated single-strand breakage, cell lethality, and the pharmacokinetics of drug uptake and efflux in human colon carcinoma cells in culture. At equivalent cytocidal concentrations, 5-iminodaunorubicin produced more single-strand breakage of DNA than Adriamycin after a 2-hr treatment interval, but the DNA scission produced by 5-iminodaunorubicin rapidly disappeared after drug removal. The kinetics of DNA breakage correlated with the rapid rates of uptake and efflux of 5-iminodaunorubicin in comparison to Adriamycin. These data emphasize the importance of the cellular pharmacokinetics of anthracyclines in relation to their cytocidal and DNA damaging properties. Moreover, the induction of equivalent single-strand breakage of DNA by similar intracellular concentrations of both drugs suggests that the free radical properties of Adriamycin are not involved in DNA scission.

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(-6) M formycin or 8-azaadenosine, but 1 to 1.4 log reductions in colony formation were produced by 10(-5) M of each analog. In the presence of 10(-6) M 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-deoxy-cytidine 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.