Determination of Pyrimidine Deoxynucleoside Triphosphates in Leukaemia Cell Extracts Containing 1-ß-D-Arabinofuranosylcytosine Triphosphate (original) (raw)
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Analytical Biochemistry, 1999
tude lower than the corresponding NTP. Hence, the quantitation of dNTP in cells is generally performed after selective oxidation or removal of the major NTP. The procedures reported so far are lengthy and cumbersome and do not enable the simultaneous determination of NTP. We report the development of a simple, direct HPLC method for the simultaneous determination of dNTP and NTP in colon carcinoma WiDr cell extracts using a stepwise gradient elution ion-pairing HPLC with uv detection at 260 nm and with a minimal chemical manipulation of cells. Exponentially growing WiDr cells were harvested by centrifugation, rinsed with phosphate-buffered saline, and carefully counted. The pellets were suspended in a known volume of ice-cold water and deproteinized with an equal volume of 6% trichloroacetic acid. The acid cell extracts (corresponding to 2.5 ؋ 10 6 cells/100 l) were centrifuged at 13,000g for 10 min at 4°C. The resulting supernatants were stored at ؊80°C prior to analysis. Aliquots (100 l) were neutralized with 4.3 l saturated Na 2 CO 3 solution prior the injection of 40 l onto the HPLC column (injection speed 250 l/min). Chromatographic separations were performed using two Symmetry C18 3.5-m (2 ؋ 3.9 ؋ 150 mm) columns (Waters), connected in series equipped with a Sentry guard column (3.9 ؋ 20 mm i.d.) filled with the same packing material. The HPLC columns were kept at 30°C. The mobile phase was delivered at a flow rate of 0.5 ml/min, with the following stepwise gradient elution program: % solvent A/solvent B, 100/0 at 0 min 3 100/0 at 1 min 3 36/64 at 5 min 3 31/69 at 90 min 3 31/69 at 105 min 3 0/100 at 106 min 3 0/100 at 120 min; 50/50 MeOH/solvent B from 121 to 130 min; 100% solvent A from 131 to 160 min. Solvent A contained 0.01 M KH 2 PO 4 , 0.01 M tetrabutylammonium chloride, and 0.25% MeOH and was adjusted to pH 7.0 (550 l 10 N NaOH for 1 liter solvent A). Solvent B consisted of 0.1 M KH 2 PO 4 , 0.028 M tetrabutylammonium chloride, and 30% MeOH and was neutralized to pH 7.0 (1.4 ml 10 N NaOH for 1 liter solvent B). Even though dNTPs are minor components of cell extracts, satisfactory regression coefficients were obtained for their calibration curves (r 2 > 0.99) established with the addition-calibration methods up to 120 pmol/40-l injection. The applicability of the method was demonstrated by in vitro studies of the modulation of NTP and dNTP pools in WiDr colon carcinoma cell lines exposed to various pharmacological concentrations of cytostatic drugs (i.e., FMdC, IUdR, gemcitabine). In conclusion, this optimized, simplified, analytical method enables the simultaneous quantitation of NTP and dNTP and may represent a valuable tool for the detection of minute alterations of cellular dNTP/NTP pools induced by anticancer/antiviral drugs and diseases.
Approach to the quantitative analysis of nucleotides by gas chromatography-mass spectromety
Journal of chromatography, 1980
Nucleotides are not ea.&y amenable to gas chromatographic analysis Cl] and for this reason the specificity and sensitrvity of gas chromatography-mass speckometry (GC-MS) with selected ion monitiring (SIM) 123 is rarely employable for nucleotide analysis. High-performance liquid chromatography (HPLC) has instead recently become a powerful and convenient method for the analysis of nucleotides 131. The specificity and sensitivi~ of HPLC analysis of nucleotides may, however, be ultimately limited since UV detection is commonly employed 141. In an effort to make nucleotides more amenable to GC-MS anaPys.is with SIM, we have devised a general scheme for the analysis of nudleosides and nucleotides (so far, ribonucleoside monophosphates) from biological media. The analytical scheme is evaluated with the analysis of nucleotide anabolites of the antineoplastic [5] and immunosuppressive 161 agent 6-mercaptopurine (G-MP). This drug must be taken up by cells and converted by hypoxanthinegutnine phos?horibosyl kansferase (HGPRT) directly into 6-mercaptqpurine riboside-$-phosphate (MPRP), a potent inhibitor of nucleic acid synthesis. MFRJ? can then be methylated in vivo at the sulfur TV give B-methyhnercaptopurine riboside-$-phosphate (MMPRP), another active anabolite. These ribo-nucIeoside monophosphates may be dephosphorylated in vivo to @ve the corresponding ribosides, 6-mercaptopurine riboside (MPR) and 6-methylmercaptopurine riboside (MMPR). This enzymatic dephosphorylation may be im-po&nt in the development of clinical resistance to 6-MP C7]-It is important,
Biochemical Pharmacology, 1988
Dinucleotides of adenosine and thymidine in the Ap.T series (n = 3,4,5 and 6) and their corresponding phosphonate analogues, where a methylene group replaces the oxygen between the alpha and beta phosphorus atoms adjacent to thymidine, have been evaluated as inhibitors of human leukaemic thymidylate kinase (dTMP kinase, EC 2.7.4.9) and ribonucleotide reductase (EC 1.17.4.1) from L1210 cells. Ap,T, Ap,T, Ap,cpT and Ap,cpT were poor inhibitors of both enzymes. Ap,T, Ap,T and their phosphonate analogues were potent inhibitors of dTMP kinase, possibly acting as bisubstrate analogues (ICKY values: Ap,T, 7.9pM; Ap,cpT, 5.8pM; Ap,T, 5.4yM; Ap,cpT, 4.0,~M). For CDP reductase, where these compounds may bridge activity/effecter sites on the Ml subunit of the enzyme, Ap,T and Ap6T were inhibitors with I& values of 14.4 PM and 20.3 PM respectively. The phosphonate series of compounds was far less active. The thymidine moiety of the compounds was essential for inhibition since Ap,A was inactive against both enzymes. dTTP, although a poor inhibitor of thymidylate kinase was a potent negative effector of CDP reductase (lqo, 19.3 PM). Significantly, Ap,T was not hydrolysed to release dlTP under the conditions of the assay. These studies show that the activities of both enzymes may be modulated by nucleotide analogues.
Analytical Biochemistry, 1999
In this paper, we describe an improved enzymatic assay for the determination of deoxyribonucleoside triphosphates (dNTPs). This is based on the elongation of 32 P 5-end-labeled oligonucleotide primers annealed to complementary oligonucleotide templates. Incorporation within the primer/template (p/t) was catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I under conditions where the concentration of the dNTP to be analyzed is limiting. Using a combination of two different sized p/t pairs, dCTP and dTTP (or dATP and dGTP) were assayed together. Since the elongated products were clearly separated after electrophoresis on a denaturing 10% polyacrylamide gel, the two dNTPs could be quantified in a single lane. This method allows for the first time the simultaneous determination of two pyrimidine or two purine deoxyribonucleoside triphosphates. Consequently, a large number of biological samples can be tested in a single experiment. The high sensitivity of this method enables the quantification of low concentrations of dNTPs, such as those found in resting nondividing cells. Furthermore, this new protocol is well suited for the determination of dNTPs in cells treated with the antiretroviral ddI, since the Klenow fragment has a low affinity for ddATP, the active form of ddI.
Information about the intracellular concentration of dNTPs and NTPs is important for studies of the mechanisms of DNA replication and repair, but the low concentration of dNTPs and their chemical similarity to NTPs present a challenge for their measurement. Here, we describe a new rapid and sensitive method utilizing hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for the simultaneous determination of dNTPs and NTPs in biological samples. The developed method showed linearity (R 2 > 0.99) in wide concentration ranges and could accurately quantify dNTPs and NTPs at low pmol levels. The intra-day and inter-day precision were below 13%, and the relative recovery was between 92% and 108%. In comparison with other chromatographic methods, the current method has shorter analysis times and simpler sample pretreatment steps, and it utilizes an ion-pair-free mobile phase that enhances mass-spectrometric detection. Using this method, we determined dNTP and NTP concentrations in actively dividing and quiescent mouse fibroblasts.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2000
To gain a more detailed insight into the metabolism of 2P,2P-difluoro-2P-deoxycytidine (dFdC, gemcitabine, Gemzar) and its effect on normal ribonucleotide (NTP) metabolism in relation to sensitivity, we studied the accumulation of dFdCTP and the changes in NTP pools after dFdC exposure in a panel of 21 solid tumour and leukaemia cell lines. Both sensitivity to dFdC and accumulation of dFdCTP were clearly cell line-dependent: in this panel of cell lines, the head and neck cancer (HNSCC) cell line 22B appeared to be the most sensitive, whereas the small cell lung cancer (SCLC) cell lines were the least sensitive to dFdC. The human leukaemia cell line CCRF-CEM accumulated the highest concentration of dFdCTP, whereas the non-SCLC cell lines accumulated the least. Not only the amount of dFdCTP accumulation was clearly related to the sensitivity for dFdC (R = 30.61), but also the intrinsic CTP/UTP ratio (R = 0.97). NTP pools were affected considerably by dFdC treatment: in seven cell lines dFdC resulted in a 1.7-fold depletion of CTP pools, in two cell lines CTP pools were unaffected, but in 12 cell lines CTP pools increased about 2-fold. Furthermore, a 1.6^1.9-fold rise in ATP, UTP and GTP pools was shown in 20, 19 and 20 out of 21 cell lines, respectively. Only the UTP levels after treatment with dFdC were clearly related to the amount of dFdCTP accumulating in the cell (R = 0.64 (P 6 0.01)), but not to the sensitivity to dFdC treatment. In conclusion, we demonstrate that besides the accumulation of dFdCTP, the CTP/UTP ratio was clearly related to the sensitivity to dFdC. Furthermore, the UTP levels and the CTP/UTP ratio after treatment were related to dFdCTP accumulation. Therefore, both the CTP and UTP pools appear to play an important role in the sensitivity to dFdC. ß
Journal of Medicinal Chemistry, 1999
Continuing our studies on ribonucleotide reductase (RNR) mechanism-based inhibitors, we have now prepared the diphosphates (DP) of 2′-O-allyl-1-D-arabinofuranosyl-uracil and-cytosine and 2′-O-allyl-9-D-arabinofuranosyl-adenine and evaluated their inhibitory activity against recombinant murine RNR. 2′-O-Allyl-araUDP proved to be inhibitory to RNR at an IC 50 of 100 µM, whereas 2′-O-allyl-araCDP was only marginally active (IC 50 1 mM) and 2′-Oallyl-araADP was completely inactive. The susceptibility of the parent nucleosides to phosphorylation by thymidine kinase and 2′-deoxycytidine kinase was also investigated, and all nucleosides proved to be poor substrates for the above-cited kinases. Moreover, prodrugs of 2′-O-allyl-araU and-araC monophosphates, namely 2′-O-allyl-5′-(phenylethoxy-L-alanyl phosphate)-araU and-araC, were prepared and tested against tumor cell proliferation but proved to be inactive. A molecular modeling study has been conducted in order to explain our results. The data confirm that for both the natural and analogue nucleoside diphosphates, the principal determinant interaction with the active site of RNR is with the diphosphate group, which forms strong hydrogen bonds with Glu623, Thr624, Ser625, and Thr209. Our findings indicate that the poor phosphorylation may represent an explanation for the lack of marked in vitro cytostatic activity of the test compounds.