PubMed Health⌕ Search

Biomedical subjects

J G Cory

Publications and source records attributed to J G Cory.

At least 37 records · Page 2Linked to original sources
PubMed1994

2'-Deoxy-2'-methylene derivatives of adenosine, guanosine, tubercidin, cytidine and uridine as inhibitors of L1210 cell growth in culture.

The 2'-deoxy-2'-methylene derivatives of adenosine (MdAdo), guanosine (MdGuo), tubercidin (MdTu), cytidine (MdCyd) and uridine (MdUrd) were synthesized as mechanism-based inhibitors directed at ribonucleotide reductase. It was shown that MdCyd 5'-diphosphate irreversibly inactivated ribonucleotide reductase from Escherichia coli (Baker et al., J Med Chem 34: 1879-1884, 1991). In studies reported here, MdAdo/EHNA, MdGuo and MdCyd inhibited L1210 cell growth with IC50 values of 3.4, 10.6 and 1.4 microM, respectively. Since MdAdo is a substrate for adenosine deaminase, the presence of EHNA was required to give maximal growth inhibition. 8-Aminoguanosine was not required to maximize the cytotoxic effects of MdGuo. The 2'-deoxy-2'-methylene derivatives of tubercidin and uridine did not inhibit L1210 cell growth at concentrations as high as 50 microM (MdTu) or 100 microM (MdUrd). L1210 cell lines resistant to hydroxyurea (directed at the non-heme iron subunit of ribonucleotide reductase) or deoxyadenosine (directed at the effector binding subunit of ribonucleotide reductase) were not resistant to MdCyd. An L1210 cell line that was highly resistant to dGuo due to the loss of a relatively specific deoxyribonucleoside kinase (Cory et al., J Biol Chem 268: 405-409, 1993) had a 6.6-fold increase in the IC50 value toward MdCyd, but showed only a 2-fold increase in resistance to MdGuo. Another L1210 cell line that was markedly deficient in adenosine kinase activity was highly resistant to MdAdo. Analysis by flow cytometry showed that MdCyd showed the transit of the cells through the G2/M phase of the cell cycle resulting in the buildup of the G2/M population. MdAdo, MdGuo and MdCyd inhibited the incorporation of [14C]cytidine into DNA without an effect on RNA synthesis or total cellular uptake of [14C]cytidine. The conversion of [14C]cytidine to deoxycytidine nucleotides was partially inhibited by MdGuo, but not by MdAdo or MdCyd. These data show that the 2'-deoxy-2'-methylene derivatives of adenosine, guanosine and cytidine are activated via specific nucleoside kinases and that the modes of action of these compounds are not identical.

Animals↗
PubMed1994

Use of nucleoside kinase-deficient mouse leukemia L1210 cell lines to determine metabolic routes of activation of antitumor nucleoside analogs.

Mouse leukemia L1210 cell lines that were selected for resistance to deoxyguanosine (dGuo-R) or lacked adenosine kinase activity (ED2) were used to evaluate the nature of the nucleoside kinase that was required to phosphorylate nucleoside analogs to their respective active nucleotide form. The dGuo-R cells had reduced levels of kinase activity toward araC, dGuo and 2-CldAdo as substrates with essentially no loss of activity toward dCyd. This cell line showed resistance to dGuo, araC, araG, FdAdo, and Fara A but not to dAdo or araA. The ED2 cell line was resistant to pyrazofurin and 6-methylmercaptopurine riboside and to araA/EHNA but not to 2-CldAdo or 2-Cl-2'-FaraA. The study of the effects of newer nucleoside analogs such as dFdCyd, MdAdo, MdCyd and MdGuo in these cell lines showed that some of these agents are primarily phosphorylated by deoxyribonucleoside kinase (dFdCyd) or by adenosine kinase (MdAdo) or in some instances by multiple kinases (FaraA). These cell lines will be useful in defining the nature of the kinase(s) responsible for activating new nucleoside analogs and defining cross-resistance patterns.

Adenosine↗
PubMed1993

Deoxyguanosine-resistant leukemia L1210 cells. Loss of specific deoxyribonucleoside kinase activity.

A mouse leukemia L1210 cell line was selected for resistance to deoxyguanosine. The deoxyguanosine-resistant cells (dGuo-R) were 126-fold less sensitive to deoxyguanosine than the wild-type cells. The IC50 values for araC and araG were increased, but only 10-12-fold in the dGuo-R cells when compared with the wild-type cells. The dGuo-R cell line showed an increased level of resistance to 2-fluoro-2'-deoxyadenosine and 2-fluoroadenine arabinoside (11-14-fold), but essentially no increase in resistance to deoxyadenosine or adenine arabinoside. Deoxyribonucleoside kinase activity was decreased only slightly (19%) when deoxycytidine was utilized as substrate; when cytosine arabinoside or deoxyguanosine was used as the substrate, the kinase activity in the extracts from the dGuo-R cells was only 10% of the enzyme activity in the extracts from the wild-type cells. The determination of the kinetic parameters, Km and Vmax, indicated that there were marked decreases in the Vmax values for deoxyguanosine and cytosine arabinoside as substrates, but not for deoxycytidine as substrate; the Km values for deoxycytidine and cytosine arabinoside were increased in the extracts from the dGuo-R cells. By use of high-performance liquid chromatography, the kinase activities in the extracts from the wild-type and resistant cells could be resolved. There was the specific loss of kinase activity toward cytosine arabinoside and deoxyguanosine as substrates. These data indicate that the dGuo-R cells have decreased levels of a specific deoxyribonucleoside kinase activity.

Animals↗
PubMed1993

Structural aspects of N-hydroxy-N'-aminoguanidine derivatives as inhibitors of L1210 cell growth and ribonucleotide reductase activity.

Previous studies have shown that N-hydroxy-N'-aminoguanidine (HAG) derivatives [RCH = NNHC(= NH)NHOH-tosylate] inhibit ribonucleotide reductase activity and block the growth of leukemia L1210 cells and human colon carcinoma, HT-29, cells in culture. In the current studies, the role of the side chains and the location of the bond of the side chain moiety to HAG were investigated using a new series of HAG derivatives which contained as the R-group--cyclohexyl, phenyl-, pyridyl- or napthyl moieties. The effects of these compounds as inhibitors of L1210 cell growth and ribonucleotide reductase activity were compared with the parent compound. N-hydroxy-N'-aminoguanidine was less inhibitory to ribonucleotide reductase activity and L1210 cell growth than hydroxyurea. The phenyl-HAG compounds which included 1-benzyloxybenzylidene- and 4-cyclohexylmethoxybenzylidene-HAG inhibited CDP reductase with IC50s which ranged from 50-110 microM. 1-Naphthylmethylene-HAG was more inhibitory than 2-naphthylmethylene-HAG and more inhibitory than the phenyl-HAG compounds. 2-Pyridylmethylene-HAG was more inhibitory than 3-pyridylmethylene- or 4-pyridylmethylene-HAG. While HAG inhibited CDP and ADP reductase activities essentially to the same extent, the HAG-derivatives inhibited ADP reductase activity to a greater extent than CDP reductase activity. Cyclohexylmethylene-HAG did not inhibit either L1210 cell growth or ribonucleotide reductase activity. There was good correlation between the inhibition of ribonucleotide reductase activity and L1210 cell growth by these HAG-derivatives. These data indicate that not only is the nature of the side chain substitution important, but also the location of the HAG-moiety on the ring position.

Animals↗
PubMed1993

Effects of 2',2'-difluorodeoxycytidine (Gemcitabine) on wild type and variant mouse leukemia L1210 cells.

2',2'-Difluorodeoxycytidine (Gemcitabine, dFdCyd) is a cytotoxic agent which is active toward a variety of tumor cells. It has been shown that there are multiple intracellular sites of action which include ribonucleotide reductase and DNA polymerase. In these studies, the effects of dFdCyd on wild-type mouse leukemia L1210 cells and variant L1210 cell lines which had alterations at the ribonucleotide reductase site or at the deoxyribonucleoside kinase site were studied. For cell growth, the IC50 value for dFdCyd in wild-type L1210 cells was 3.1 nM. In the variant cell lines, the IC50 values were: hydroxyurea-resistant (HU), 3.3 nM; deoxyadenosine-resistant (Y8), 1.8 nM; pyrazoloimidazole/deoxyadenosine-resistant (ED2), 1.9 nM; and deoxyguanosine-resistant (dGuo-R), 44.7 nM. The dGuo-R cell line had a relatively specific loss of the deoxyribonucleoside kinase responsible for phosphorylating deoxyguanosine and cytosine arabinoside with little loss of the deoxycytidine kinase activity. DFdCyd had no effect on the total uptake of [14C]cytidine into the cells or incorporation into RNA. DFdCyd inhibited the conversion of [14C]cytidine to deoxycytidine nucleotides and incorporation into DNA. However, the incorporation of cytidine into DNA was inhibited to a greater extent than was the inhibition of in situ ribonucleotide reductase activity. Ribonucleotide reductase activity in cell-free extracts prepared from L1210 cells treated with dFdCyd (20 nM) overnight was reduced by 50%. These results show that cell lines which have increased levels of ribonucleotide reductase activity (HU and ED2) or loss of feedback inhibition by dATP (ED2 and Y8) are still sensitive to dFdCyd. The findings indicate that ribonucleotide reductase is not the primary site of inhibition by dFdCyd.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Altered steady-state levels of the messenger RNAs for c-myc and p53 in L1210 cell lines resistant to deoxyadenosine.

L1210 cell lines, selected for resistance to deoxyadenosine due to the loss of allosteric inhibition of ribonucleotide reductase by dATP, had altered steady-state levels of the mRNAs for c-myc, fos, and p53. Wild-type L1210 cells had constitutive steady-state levels of c-myc and p53 with little or no fos mRNA. Two different deoxyadenosine-resistant cell lines (Y8 and ED2) had elevated steady-state levels of c-myc and fos but essentially no p53 mRNA. Hydroxyurea-resistant L1210 cells had the same levels of c-myc, fos, and p53 as the wild-type cells. There was no amplification of the gene for c-myc in the Y8 or ED2 cell lines. The half-life for c-myc mRNA was essentially the same in the wild-type and the Y8 and ED2 cells. Nuclear runoff experiments showed that the rates of transcription for c-myc in the Y8 and ED2 cells were elevated and could account for the increased steady-state levels of c-myc in these two cell lines. The transcription rate for p53 mRNA was not decreased in the Y8 and ED2 cells and therefore did not account for the loss of the steady-state levels of p53 in the cells. Cycloheximide treatment of the Y8 and ED2 cells resulted in a marked increase in the steady-state p53 mRNA level, indicating that a protein which was rapidly turned over was responsible for the extremely short half-life of p53 mRNA in these two cell lines.

Animals↗
PubMed1992

Factors affecting the mRNA levels for the non-heme iron and effector-binding subunits of ribonucleotide reductase.

Ribonucleotide reductase which catalyzes the rate-limiting step in the de novo synthesis of dNTPs is composed of two non-identical protein subunits which are not under coordinate control in terms of synthesis and degradation. The mRNAs for the effector-binding (EB) and non-heme iron (NHI) subunits are likewise not under coordinate control during cell cycle traverse. Inhibitors directed at the specific subunits of ribonucleotide reductase block DNA synthesis. These current studies show that drugs such as IMPY or hydroxyurea which specifically inhibit the NHI subunit cause a marked increase in the steady-state level of the mRNA for the NHI subunit while resulting in a decrease in the level of mRNA for the EB subunit. In cells treated with deoxyadenosine, the patterns of the mRNAs for the NHI and EB subunits were different from those seen in the IMPY- or hydroxyurea-treated cells. Control experiments utilizing inhibitors (aphidicolin or araC) directed at DNA polymerase showed that the pattern of changes in the mRNA levels for the NHI and EB subunits were specific for the reductase inhibitors. These changes in the mRNAs for the NHI and EB subunits may be due to drug-induced alterations in transcription rates and/or degradation rates for the specific mRNAs.

Animals↗
PubMed1992

5-hexyl-2'-deoxyuridine inhibition of nucleoside transport in L1210 cells.

Previous studies have shown that 5-hexyl-2'-deoxyuridine (HdUrd) blocked the cytotoxic effects of 5-fluorodeoxyuridine and deoxyadenosine in L1210 cells. HdUrd had no effect in preventing the inhibitory effects of 5-fluorouracil. These data suggested that HdUrd was an inhibitor of nucleoside transport in L1210 cells (Cory, J. G.; Halley, M. C.; Janey, A.; Lapis, K. Cancer Res. 50:4552-4556; 1990). Studies have now been carried out which show that HdUrd inhibits nucleoside transport as measured by [3H]uridine or [3H]formycin B transport into L1210 cells in culture. The IC50 for HdUrd inhibition of total [3H]uridine uptake was approximately 20 microM in wild-type L1210 cells. Since wild-type L1210 cells have three distinct nucleoside transporters, the effect of HdUrd on each transporter was examined using the non-metabolized nucleoside analog, formycin B. The nitrobenzylmercaptopurine riboside (NBMPR)-sensitive transporter, es, was most sensitive to HdUrd with an IC50 of 1.0 +/- 0.1 microM; the NBMPR-insensitive transporter, ei, was much less sensitive to HdUrd with an IC50 of 32 +/- 2 microM; the sodium ion-dependent transporter, cif, was the least sensitive transporter to HdUrd with an IC50 of 130 +/- 5 microM. These data support the concept that HdUrd, a relatively non-cytotoxic agent, could be useful in increasing the potency of antitumor inhibitors directed at the de novo pathways for nucleotide synthesis through the blockage of the salvage pathways for nucleosides.

Animals↗
PubMed1991

Studies on the mechanisms of inhibition of L1210 cell growth by 3,4-dihydroxybenzohydroxamic acid and 3,4-dihydroxybenzamidoxime.

Didox and Amidox inhibit L1210 cell growth in culture. At least one of the mechanisms in the mode(s) of action of the compounds is directed at the ribonucleotide reductase site. Partially purified preparations of ribonucleotide reductase activity are inhibited by Amidox and Didox. The formation of deoxycytidine nucleotides from [14C]cytidine in intact L1210 cells is also blocked. Didox and Amidox cause the decrease in the intracellular pools of the four dNTPs. Hydroxyurea-resistant L1210 cells are not cross-resistant to either Didox or Amidox. These data suggest that Didox and Amidox are not inhibiting ribonucleotide reductase through a mechanism similar to hydroxyurea.

Animals↗
PubMed1991

Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture.

A new tetrazolium analog of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was evaluated as a substitute for MTT in the microculture screening assay for in vitro cell growth. This new tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), in the presence of phenazine methosulfate (PMS), gave a water-soluble formazan product that had an absorbance maximum at 490-500 nm in phosphate-buffered saline. The amount of colored product formed was proportional to the number of cells and the time of incubation of the cells with MTS/PMS. MTS/PMS was reactive in all the cell lines tested which included mouse leukemia L1210 cells, mouse Ehrlich tumor cells, mouse 3T3 fibroblasts, and human colon tumor cells (HT-29). HT-29 and 3T3 fibroblasts reduced MTS/PMS more efficiently than they reduced MTT. Comparable to the amount of product formed from MTT, MTS/PMS gave excellent product formation. The IC50 value for pyrazoloimidazole obtained using MTS/PMS was 200 microM; for 5-fluoro-2'-deoxyuridine, the IC50 value was 0.9 nM. These values compared very favorably with the IC50 values obtained by direct cell counts. Further, the same IC50 values were obtained when the absorbances of the formazan product in the 96-well plates were determined after different times of incubation.

Animals↗
PubMed1991

Cell-cycle associated transcriptional regulation of ribonucleotide reductase in L1210 leukemia cells and drug-resistant variants.

Previous studies from this laboratory have shown that the steady-state levels of the mRNA for the non-heme iron (NHI) subunit of ribonucleotide reductase were markedly elevated in hydroxyurea-resistant L1210 cell lines with minimal changes in the mRNA levels for the effector-binding (EB) subunit. In the present study, wild-type L1210 cells and their drug-resistant variants [hydroxyurea-resistant (HU-7); deoxyadenosine-resistant (Y-8); and deoxyadenosine/pyrazoloimidazole-resistant (ED2)] were synchronized by EGTA treatment in the G0/G1-phase of the cell cycle. Upon the addition of CaCl2, the cells reentered the cell cycle. The steady-state levels and the transcriptional rates of the mRNAs for the EB subunit and glyceraldehyde-3-phosphate dehydrogenase were measured and found to be similar in the drug-resistant variants compared to the wild-type cells. While the steady-state level of the mRNA for the NHI subunit was increased 35-fold in the HU-7 cell line, the transcription rate was increased only 7-fold. The increase in the transcription rate did not account for the large increase in the steady-state level. These data indicate that the increased steady-state level of the mRNA for the NHI subunit in the HU-7 L1210 cell line was not due to cell-cycle differences and that post-transcriptional processing and/or stability may play a role as well.

Animals↗
PubMed1990

Inhibition of ribonucleotide reductase and growth of human colon carcinoma HT-29 cells and mouse leukemia L1210 cells by N-hydroxy-N'-aminoguanidine derivatives.

A series of N-hydroxy-N'-aminoguanidine (HAG) derivatives were studied and compared for their effects on ribonucleotide reductase activity in cell-free extracts; on nucleic acid synthesis and the growth of human colon carcinoma HT-29 cells; and on mouse leukemia L1210 cells in culture. The HAG derivatives [RCH=NNHC(=NH)NHOH-tosylate] studied could be grouped as: (1) hydroxybenzylidines; (2) methoxybenzylidines; and (3) nitrobenzylidines substituted at the R position. 2'-Hydroxybenzylidine-HAG, the lead compound, was relatively active in both HT-29 cells and L1210 cells (20 +/- 5 and 13 +/- 4 microM for 50% inhibition of HT-29 and L1210 cell growth respectively). The monohydroxybenzylidene compounds were generally more active than the dihydroxy- and trihydroxybenzylidene-HAG derivatives. The methoxybenzylidene-HAGs were as active as the monohydroxybenzylidene-HAGs. 2'-Hydroxy-4'-methoxybenzylidene-HAG was much more active than 2',4'-dihydroxybenzylidene-HAG. The mononitrobenzylidene-HAGs were more active than the dinitrobenzylidene-HAG compound. In general, L1210 cells were more sensitive to the effects of the HAG compounds than were HT-29 cells. There was good agreement between the concentration of drug required to inhibit the growth of HT-29 cells and that required to inhibit the growth of L1210 cells. There was also good correlation between the ability of HAG derivatives to inhibit ribonucleotide reductase activity and to inhibit tumor cell growth. Some derivatives, such as 2',3',4'- and 3',4',5'-trihydroxybenzylidene-HAG inhibited L1210 cell growth by 50% at lower concentrations (7.8 and 11.9 microM respectively) than the concentrations needed for 50% inhibition of HT-29 cell growth (196 and 234 microM respectively) and ribonucleotide reductase activity (122 and 188 microM respectively). The studies of nucleic acid synthesis in L1210 cells using [3H]cytidine as a precursor showed that 2',3',4'-trihydroxybenzylidine-HAG inhibited DNA synthesis at a lower concentration (29 microM for 50% inhibition) than was needed for the inhibition of RNA synthesis and formation of [3H]deoxycytidine nucleotides in the acid-soluble fraction (320 and 820 microM for 50% inhibition respectively). These results indicate that 2',3',4'-trihydroxybenzylidine-HAG inhibits DNA synthesis in L1210 cells through other mechanisms rather than exclusively through the inhibition of ribonucleotide reductase activity.

Animals↗
PubMed1990

5-Hexyl-2'-deoxyuridine blocks the cytotoxic effects of 5-fluorodeoxyuridine or deoxyadenosine in leukemia L1210 cells in culture.

Antitumor agents which block the de novo synthesis of nucleotides can be circumvented by the presence of salvage pathways for the reutilization of nucleobases and nucleosides. Studies have been carried out which show that 5-hexyl-2'-deoxyuridine (HdUrd) effectively blocks the cytotoxic effects of deoxyadenosine and fluorodeoxyuridine in L1210 cells. Although HdUrd (500 microM) had essentially no effect on the growth of L1210 cells in culture, the total uptake of [14C]cytidine into these cells was inhibited 99% by this concentration of HdUrd. The inhibitory effects of fluorodeoxyuridine (FdUrd) and deoxyadenosine could be completely prevented by the presence of HdUrd (200 microM). The growth inhibitory effects of fluorouracil were not prevented by HdUrd. Dipyridamole prevented the inhibition of L1210 cell growth by FdUrd but not by deoxyadenosine or fluorouracil. 5-Isopropyl-, 5-pentyl-, and 5-octyldeoxyuridine were not effective in preventing the cytotoxic effects of deoxyadenosine. The data suggest that HdUrd might be useful in blocking the salvage of nucleosides, thereby potentiating the effects of inhibitors of de novo nucleotide synthesis.

Animals↗
PubMed1990

Effect of ribonucleotide reductase inhibitors on the growth of human colon carcinoma HT-29 cells in culture.

The effects of ribonucleotide reductase inhibitors on the growth of the human colon carcinoma cell line HT-29 were examined. Inhibitors were chosen for these studies that were specifically directed at each of the subunits of ribonucleotide reductase. The concentrations of drugs required to inhibit the growth of HT-29 cells by 50% (IC50) for hydroxyurea, 2,3-dihydro-lH-pyrazole-[2,3a]imidazole (IMPY), and 4-methyl-5-amino-l-formyl-isoquinoline thiosemicarbazone (MAIQ) were 206, 996, and 3.2 microM, respectively. Although the IC50 for deoxyadenosine alone was greater than 2,000 microM, in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), which protects deoxyadenosine from deamination by adenosine deaminase, it was reduced to 112 microM. The IC50 for deoxyguanosine was 1,060 microM. The addition of 8-aminoguanosine to protect deoxyguanosine from phosphorolysis by purine nucleoside phosphorylase did not increase the toxicity of deoxyguanosine in HT-29 cells. The combination of MAIQ or IMPY and deoxyadenosine/EHNA gave strong synergistic inhibition of HT-29 cell growth. The results of these studies indicate that ribonucleotide reductase inhibitors effectively block the growth of human colon carcinoma HT-29 cells and that combinations of inhibitors directed at the individual subunits of reductase result in synergistic inhibition of HT-29 cell growth in culture.

Adenine↗
PubMed1990

Effects of cytosine arabinoside and hydroxyurea on the synthesis of deoxyribonucleotides and DNA replication in L1210 cells.

Experiments were carried out in L1210 cells to examine the importance of 'substrate cycles' in regulating the intracellular levels of deoxyribonucleoside 5'-triphosphate. L1210 cells were incubated with [14C]cytidine or [14C]adenosine in the presence and absence of hydroxyurea or cytosine arabinoside (araC). These incubations were carried out for either 30 or 120 min. Inhibition of ribonucleotide reductase by hydroxyurea resulted in the blockage of the flux of ribonucleotides to deoxyribonucleotides (greater than 90%) as expected. When DNA synthesis was inhibited with araC, there was a marked decrease in the incorporation of [14C]cytidine or [14C]adenosine into DNA as deoxyribonucleotides. However, there was not a corresponding increase in the deoxyribonucleotide levels in the acid-soluble fraction or deoxyribonucleosides in the culture medium. AraC treatment decreased the total formation of deoxyribonucleotides. These data indicate that L1210 cells do not regulate the intracellular pools of dNTPs via 'substrate cycles' which involve activation of phosphatases when DNA synthesis is blocked or activation of kinases when ribonucleotide reductase is inhibited.

Adenosine↗
PubMed1990

Antineoplastic effect of the combination of 2,3-dihydro-1H-pyrazole[2,3a]imidazole plus deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine in mice with L1210 leukemia cells.

Administration of 2,3-dihydro-1H-pyrazole[2,3a]imidazole (IMPY, 150 mg/kg) followed 8 hr later by injection of deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine (dAdo/EHNA, 175 mg/17.5 mg/kg) on days 2, 3, 6, and 7 increased the mean survival time of L1210 tumor bearing mice (210%). The sequential treatment was more efficacious than the simultaneous administration of these drugs. Administration of IMPY or dAdo/EHNA, alone, at the same doses as in the combination, did not prolong the life-span of tumor bearing mice. To determine the basis for the increased survival due to the sequential treatment with IMPY and dAdo/EHNA, cell cycle analysis and deoxyribonucleoside triphosphate concentrations were measured. Cytotoxicity of IMPY and dAdo/EHNA is known to be achieved through the inhibition of ribonucleotide reductase. IMPY is a specific inhibitor of the nonheme-iron subunit of ribonucleotide reductase, whereas deoxyadenosine in the presence of the adenosine deaminase inhibition, EHNA, is converted to deoxyadenosine 5'-triphosphate (dATP), which is a specific inhibitor of the effector-binding-subunit of ribonucleotide reductase. Our studies showed that L1210 cells accumulated in early S-phase, whereas intracellular dATP and deoxyguanosine triphosphate (dGTP) pools were depleted 8 hr after IMPY administration. dAdo/EHNA administration 8 hr after IMPY injection caused an increase in the intracellular concentration of dATP while maintaining the depletion of the dGTP pool and prolonged the S-phase as compared to the administration of IMPY alone.

Adenine↗
PubMed1989

Metabolic activation of 2,6-diaminopurine and 2,6-diaminopurine-2'-deoxyriboside to antitumor agents.

2,6-Diaminopurine (DAP) and 2,6-diaminopurine 2'-deoxyriboside (DAPdR) are analogs of adenine and deoxyadenosine, respectively. It was the purpose of this study to compare these analogs under identical conditions in order to define their inhibitory properties and the underlying mechanism in L1210 mouse leukemia cells. In a 5-day cell growth experiment, DAP exerted a significantly stronger antiproliferative effect than DAPdR. Correspondingly, colony formation of L1210 cells in soft agarose was inhibited by DAP to a greater extent than by DAPdR. A differential distribution of L1210 cells in the cell cycle resulted from an exposure to DAP and DAPdR. While DAPdR arrested cells in the G1/G0 phase of the cell cycle, DAP appeared to lead to an accumulation of G2/M cells. The diaminopurines were combined with modulatory agents to test the antiproliferative action of the combinations. Deoxycytidine partially rescued the cells from the growth inhibitory action of DAPdR without affecting the growth of DAP-treated cells. When adenine was used, the antiproliferative effect of DAPdR was slightly enhanced while the effect of DAP was completely abolished. 8-Aminoguanosine, a specific inhibitor of purine nucleoside phosphorylase, synergistically potentiated the cytostatic effect of DAPdR. However, this inhibitor did not alter DAP effects. At the biochemical level, the target of DAPdR was ribonucleotide reductase which was in line with a drastic expansion of the dGTP pool in DAPdR-treated cells. In cells exposed to DAP, high levels of DAP riboside triphosphate were measured; concomitantly, the ATP level dropped markedly. Enzymological studies revealed that DAPdR is an excellent substrate of adenosine deaminase giving rise to the formation of deoxyguanosine. DAP was found to be activated in the purine nucleoside phosphorylase reaction and in a phosphoribosyl-pyrophosphate-dependent reaction. The data from this comparative study suggest that DAPdR and DAP possess different toxicity mechanisms. DAPdR and DAP possess different toxicity mechanisms. DAPdR acts as a precursor of deoxyguanosine, and DAP is metabolically activated to DAP-containing ribonucleotide analogs. These different metabolic routes seem to account for the different effects of DAP and DAPdR at the cellular level.

Adenosine↗