scholarly journals Guanine for DNA synthesis. A compulsory route through ribonucleotide reductase

1988 ◽  
Vol 255 (3) ◽  
pp. 1045-1048 ◽  
Author(s):  
D S Duan ◽  
W Sadee
Keyword(s):  
Dna Synthesis ◽  
Cell Line ◽  
Ribonucleotide Reductase ◽  
Purine Nucleoside Phosphorylase ◽  
S Phase ◽  
Alternative Pathways ◽  
Dose Dependent Fashion ◽  
T Lymphoma ◽  
Dose Dependent ◽  
Tracer Experiments

Two alternative pathways for the synthesis of dGTP and its incorporation into DNA were studied: guanine (Gua)→GMP→GDP→dGDP→dGTP→DNA and dG→dGMP→dGDP→dGTP→DNA. To determine the contribution of each pathway to DNA synthesis independently of each other, [14C]Gua and [3H]dG tracer experiments were performed in a double-mutant S-49 mouse T-lymphoma cell line, dGuo-L, with purine nucleoside phosphorylase (EC 2.4.2.1)-deficiency and dGTP-feedback-resistant ribonucleotide reductase (RR, EC 1.17.4.1). In this cell line, dGTP pools can be selectively elevated by exogenous dG without affect RR and DNA synthesis. Although [3H]dG, but not [14C]Gua (up to 200 microM), readily expanded the cellular dGTP pool in a dose-dependent fashion in asynchronous cells, only a small fraction of the Gua flux into DNA was derived from [3H]dG, with the major fraction coming from [14C]Gua. H.p.l.c. analysis of G1- and partially enriched S-phase cells revealed that [3H]dGTP only accumulates in G1- but not in S-phase cells because of a rapid turnover of the dGTP pool during DNA synthesis. These results fail to provide evidence for cellular dGTP compartmentation and suggest that the pathway dG→dGMP→dGDP→dGTP alone has insufficient capacity to maintain DNA synthesis.

scholarly journals Mechanisms of 2′-deoxyguanosine toxicity in mouse T-lymphoma cells with purine nucleoside phosphorylase deficiency and resistance to inhibition of ribonucleotide reductase by dGTP

1990 ◽  
Vol 268 (3) ◽  
pp. 725-731 ◽  
Author(s):  
D S Duan ◽  
T Nagashima ◽  
T Hoshino ◽  
F Waldman ◽  
K Pawlak ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Double Mutant ◽  
Purine Nucleoside Phosphorylase ◽  
Feedback Inhibition ◽  
S Phase ◽  
G1 Phase ◽  
Wild Type ◽  
L Cells ◽  
T Lymphoma

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency is thought to cause T-lymphocyte depletion by accumulation of dG and dGTP, resulting in feedback inhibition of ribonucleotide reductase (RR; EC 1.17.4.1) and hence DNA synthesis. To test for additional toxic mechanisms of dG, we selected a double mutant of the mouse T-lymphoma S-49 cell line, dGuo-L, which is deficient in PNP and partially resistant to dGTP feedback inhibition of RR. The effects of dG on dGuo-L cells (concn. causing 50% inhibition, IC50 = 150 microM) were compared with those on the wild-type cells (IC50 = 30 microM) and the NSU-1 mutant with PNP deficiency only (IC50 = 15 microM). Fluorescence flow cytometry showed that equitoxic dG concentrations arrested wild-type and NSU-1 cells at the G1-S interface while allowing continued DNA synthesis in the S-phase, whereas the double mutant dGuo-L cells progressed through the cell cycle normally. dGuo-L cells accumulated high levels of dGTP in G1-phase, but not in S-phase cells, because of the utilization of dGTP for DNA synthesis and limited capacity to synthesize dGTP from dG. These results support the hypothesis that dG/dGTP toxicity occurs in the G1-phase or at the G1-S interface. Failure of dG to arrest the double mutant dGuo-L cells at the G1-S interface allows these cells to escape into S-phase, with an accompanying drop in dGTP levels. Thus the partial resistance of dGuo-L cells to dG toxicity may result from their shorter residence time in G1, allowing them to sustain higher dGTP levels. Hence RR inhibition by dGuo may not be the primary toxic mechanism in S-49 cells; rather, it may serve as an accessory event in dG toxicity by keeping the cells in the sensitive phase of the cell cycle. Among the possible targets of dG toxicity is RNA synthesis, which was inhibited at an early stage in dGuo-L cells.

scholarly journals Growth factors, signaling pathways, and the regulation of proliferation and differentiation in BC3H1 muscle cells. I. A pertussis toxin-sensitive pathway is involved.

1989 ◽  
Vol 108 (1) ◽  
pp. 159-167 ◽  
Author(s):  
D J Kelvin ◽  
G Simard ◽  
H H Tai ◽  
T P Yamaguchi ◽  
J A Connolly
Keyword(s):  
Growth Factors ◽  
Cell Line ◽  
Pertussis Toxin ◽  
Northern Analysis ◽  
Skeletal Muscle Myosin ◽  
Proliferation And Differentiation ◽  
Dose Dependent Fashion ◽  
Camp Levels ◽  
Dose Dependent ◽  
In Cells

Cells of the nonfusing muscle cell line BC3H1 stop proliferating and express a family of muscle-specific proteins when the FBS concentration is reduced from 20 to 0.5% (Munson, R., K.L. Caldwell, and L. Glaser. 1982. J. Cell Biol. 92:350-356). Several growth factors have been shown to block differentiation in this cell line. To begin to investigate the potential role of G proteins in signal transducing pathways from these receptors, we have examined the effects of cholera toxin (CT) and pertussis toxin (PT) on proliferation and differentiation in BC3H1 cells. PT specifically ADP ribosylates a protein with an apparent molecular mass of 40 kD in BC3H1 cell membranes, whereas CT specifically ADP ribosylates three proteins of 35-43 kD. When added to exponentially growing cells in 20% FBS, CT and PT inhibited [3H]thymidine incorporation by up to 75% in a dose-dependent fashion. We found the synthesis of creatine kinase (CK) and skeletal muscle myosin light chain was reversibly induced in cells in 20% FBS treated with PT, but no increased synthesis was seen in cells treated with CT or in control cells; Northern analysis indicated this induction was at the level of mRNA. In cells shifted to 0.5% FBS, CT inhibited the normally induced synthesis of CK whereas PT potentiated it by approximately 50%. Forskolin also inhibited growth in 20% FBS and differentiation in 0.5% FBS medium in a dose-dependent fashion. both forskolin and CT elevated cAMP levels compared with control or PT-treated cells, suggesting that CT is blocking proliferation and differentiation by elevating cAMP levels. These results establish that a PT-sensitive pathway is involved in regulating proliferation and differentiation in BC3H1 cells, and we postulate that PT functions by ADP ribosylating a G protein that transduces signals from growth factor receptors in these cells.

Resveratrol blocks interleukin-1β–induced activation of the nuclear transcription factor NF-κB, inhibits proliferation, causes S-phase arrest, and induces apoptosis of acute myeloid leukemia cells

Blood ◽  
2003 ◽  
Vol 102 (3) ◽  
pp. 987-995 ◽  
Author(s):  
Zeev Estrov ◽  
Shishir Shishodia ◽  
Stefan Faderl ◽  
David Harris ◽  
Quin Van ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
S Phase ◽  
Interleukin 1Β ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Acute Myeloid

Abstract Resveratrol, an edible polyphenolic stilbene, has been reported to possess substantial antileukemic activities in different leukemia cell lines. We investigated whether resveratrol is active against fresh acute myeloid leukemia (AML) cells and its mechanism of action. Because interleukin 1β(IL-1β) plays a key role in proliferation of AML cells, we first tested the effect of resveratrol on the AML cell lines OCIM2 and OCI/AML3, both of which produce IL-1β and proliferate in response to it. Resveratrol inhibited proliferation of both cell lines in a dose-dependent fashion (5-75 μM) by arresting the cells at S phase, thus preventing their progression through the cell cycle; IL-1β partially reversed this inhibitory effect. Resveratrol significantly reduced production of IL-1β in OCIM2 cells. It also suppressed the IL-1β–induced activation of transcription factor nuclear factor κB (NF-κB), which modulates an array of signals controlling cellular survival, proliferation, and cytokine production. Indeed, incubation of OCIM2 cells with resveratrol resulted in apoptotic cell death. Because caspase inhibitors Ac-DEVD-CHO or z-DEVD-FMK partially reversed the antiproliferative effect of resveratrol, we tested its effect on the caspase pathway and found that resveratrol induced the activation of the cysteine protease caspase 3 and subsequent cleavage of the DNA repair enzyme poly (adenosine diphosphate [ADP]–ribose) polymerase. Finally, resveratrol suppressed colony-forming cell proliferation of fresh AML marrow cells from 5 patients with newly diagnosed AML in a dose-dependent fashion. Taken together, our data showing that resveratrol is an effective in vitro inhibitor of AML cells suggest that this compound may have a role in future therapies for AML.

scholarly journals Ribonucleotide Reductase: In-vitro S-glutathionylation of R2 and p53R2 Subunits of Mammalian Class I Ribonucleotide Reductase Protein

2021 ◽  
Author(s):  
Ajanta Chatterji ◽  
Arne Holmgren ◽  
RAJIB SENGUPTA
Keyword(s):  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
S Phase ◽  
Future Research ◽  
Dna Damage And Repair ◽  
Treatment Regimes ◽  
Rate Limiting Step ◽  
Catalytic Cycles ◽  
R2 Subunit

Abstract Ribonucleotide reductases (RNR) catalyze the rate-limiting step in DNA synthesis during the S-phase of the cell cycle. Its constant activity in order to maintain dNTP homeostasis is a fascinating area of research and an attractive candidate for cancer research and antiviral drugs. Redox modification such as S-glutathionylation of the R1 subunit of mammalian RNR protein has been presumed to regulate the activity of RNR during catalytic cycles. Herein, we report S-glutathionylation of the R2 subunit. We have also shown Grx1 system can efficiently deglutathionylate the S-glutathionylated R2 subunit. Additionally, our data also showed for the very first time S-glutathionylation of mammalian p53R2 subunit that regulates DNA synthesis outside S-phase during DNA damage and repair. Taken together, these data will open new avenues for future research relating to exact physiological significance, target thiols, and/or overall RNR activity due to S-glutathionylation of R2 and p53R2 subunits and provide valuable insights for effective treatment regimes.

Inhibition of the Jak/Stat Pathway Downregulates Immunoglobulin Production and Induces Cell Death in Waldenström Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1691-1691
Author(s):  
Stephen M Ansell ◽  
Deanna Grote ◽  
Sherine F. Elsawa ◽  
Mamta Gupta ◽  
Steven C Ziesmer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Thymidine Uptake ◽  
Complete Suppression ◽  
Dose Dependent Fashion ◽  
Dose Dependent ◽  
Stat Pathway

Abstract Abstract 1691 Poster Board I-717 Waldenström macroglobulinemia (WM) is a B-cell malignancy that is characterized by the production of a monoclonal IgM protein and a lymphoplasmacytic infiltrate in the bone marrow. The aberrant production of the monoclonal IgM can result in serum hyperviscosity that can cause significant morbidity in patients with this disease. In previous work, we have shown that IL-6 significantly upregulates IgM secretion by WM cells and that IL-6 secretion is regulated by CCL5 (Rantes). We have also shown that IL-6 mediated IgM secretion in WM requires phosphorylation of Stat1 and Stat3. Because IL-6 induced signaling involves the Jak/Stat pathway, we tested whether the use of a Jak/Stat inhibitor, TG101348, would result in down regulation of CCL5, IL-6 and IgM production and inhibit cell proliferation and viability in WM. First, we determined whether TG101348 could inhibit the production of CCL5 because other Jak inhibitors have been shown to inhibit cytokine production. Using the BCWM.1 cell line as well CD19+ malignant cells from bone marrow specimens from WM patients, we measured CCL5 by ELISA in the culture supernatant 24 hours after treatment with increasing concentrations of the inhibitor. We found that CCL5 secretion was decreased by 50% at a concentration of TG101348 of 250nM and was completely inhibited at 2μM. Next, we measured IL-6 production after treatment with TG101348. We had previously shown that stromal cells are the primary source of IL-6 and therefore used the stromal cell line HS-5 to measure IL-6 by ELISA after treatment with the inhibitor. Our previous work had also shown that IL-6 secretion was mediated by GLI (a member of the Hedgehog pathway) rather than the Jak/Stat pathway. Interestingly, we found that IL-6 secretion was inhibited in a dose dependent fashion but required higher doses for complete suppression (8μM). We then measured IgM production by malignant B-cells 24 hours after treatment with TG101348. Our previous work had shown that IL-6 mediated IgM secretion was dependent on the Jak/Stat pathway. We found that IgM production was inhibited by 50% at 500nM and completely suppressed at 2μM. Finally, we measured the effect of TG101348 on cell proliferation and survival. Using the BCWM.1 cell line, we found that cell proliferation as determined by tritiated thymidine uptake was inhibited in a dose dependent fashion with 50% inhibition at 1μM. Inhibition of cell viability as measured by Annexin V/propidium iodide staining, however, required higher concentrations and cell viability was inhibited with an IC50 of 8μM. These data confirm the role of Jak/Stat signaling in the CCL5-IL-6-IgM axis in WM. We found that TG101348 generally suppressed the signaling and growth of WM cells but that pathways that were known to be Jak/Stat dependent required significantly lower doses to be completely inhibited. These data provide a strong rationale for the use of inhibitors of this pathway, such as TG101348, in the treatment of patients with WM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Early synthesis of specific cytoplasm proteins is correlated with the rate of exit of lymphocytes from the resting state.

1984 ◽  
Vol 99 (5) ◽  
pp. 1814-1821 ◽  
Author(s):  
D J Hall ◽  
J J O'Leary ◽  
A Rosenberg
Keyword(s):  
Resting State ◽  
Human Lymphocytes ◽  
Early Response ◽  
S Phase ◽  
Polyacrylamide Gels ◽  
Cytoplasmic Proteins ◽  
Cellular Aggregation ◽  
Dose Dependent Fashion ◽  
Pha Stimulation ◽  
Dose Dependent

We investigated the initiation of synthesis of proteins in human lymphocytes exposed to the mitogen phytohemagglutinin (PHA) for 6 h. Radiolabeled proteins in three subcellular fractions, cytoplasmic, nuclear salt wash, and nuclear, were separated on polyacrylamide gels. Compared with cells incubated for the same time in the absence of PHA only two cytoplasmic proteins of Mr 51 and 60 kd showed increased synthesis in a dose-dependent fashion. Synthesis of the 60-kd protein shows the strongest correlation with rate of entry into the first S phase and with rate of cellular aggregation. Thus, the 60-kd protein appears to be a major early response-associated protein for entry of lymphocytes into the first S phase after PHA stimulation.

scholarly journals Compartmentation of guanine nucleotide precursors for DNA synthesis

1986 ◽  
Vol 234 (2) ◽  
pp. 263-269 ◽  
Author(s):  
B T Nguyen ◽  
W Sadée
Keyword(s):  
Dna Synthesis ◽  
Mycophenolic Acid ◽  
Purine Nucleoside Phosphorylase ◽  
Guanine Nucleotide ◽  
Nucleotide Incorporation ◽  
T Lymphoma ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Inhibition Of Dna Synthesis ◽  
Mutant Cells ◽  
Kinetics Of

We have studied the kinetics of guanine incorporation into DNA in mouse T-lymphoma (S-49) mutant cells [PNPase (purine-nucleoside phosphorylase)- and HGPRTase (hypoxanthine: guanine phosphoribosyltransferase)-deficient] that are incapable of converting dGuo (deoxyguanosine) to Gua (guanine) ribonucleotides. Of the two possible pathways for an exogenous guanine source to reach DNA, firstly: dGuo→dGMP→dGDP→dGTP and secondly: Gua→GMP→GDP→dGDP→dGTP only the second pathway was found to be functional in providing guanine for DNA replication, although deoxyguanosine readily produced toxic cellular dGTP levels via the first pathway. The functional guanine-nucleotide-precursor pools for DNA are rather small; further, the depletion of the small GMP pool, but not that of GDP, GTP and dGTP, correlated well with the inhibition of DNA synthesis by mycophenolic acid, an IMP dehydrogenase inhibitor. These results support the hypothesis that guanine-nucleotide incorporation into DNA is highly compartmentalized and that a small functional guanine-nucleotide pool, e.g., the GMP pool, may serve a crucial role in limiting the availability of DNA precursor substrate.

Buthionine sulfoximine-induced cytostasis does not correlate with glutathione depletion

1992 ◽  
Vol 262 (1) ◽  
pp. C122-C127 ◽  
Author(s):  
Y. J. Kang ◽  
M. D. Enger
Keyword(s):  
Dna Synthesis ◽  
Rat Kidney ◽  
Buthionine Sulfoximine ◽  
Glutathione Depletion ◽  
Quiescent Cells ◽  
Dose Dependent Fashion ◽  
Forming Efficiency ◽  
Normal Rat ◽  
Epidermal Growth ◽  
Dose Dependent

The effects of L-buthionine-(S,R)-sulfoximine (BSO) on the proliferation of normal rat kidney fibroblasts (NRK-49F) were determined and compared with the effects of BSO on cellular glutathione (GSH) content. The proliferation rate of exponentially growing NRK-49F cells was found to be slowed in 0.01 and 0.1 mM BSO and arrested in 1.0 and 10 mM BSO. There is no retardation in the proliferation of cells cultured in 0.001 mM BSO. However, varying BSO concentrations at and above 0.1 mM did not result in concordant differences in the rate and extent of GSH depletion. A dose-dependent effect of BSO on GSH levels was observed at BSO concentrations less than or equal to 0.01 mM. BSO was found also to inhibit epidermal growth factor (EGF)-induced DNA synthesis in NRK-49F cells arrested by serum deprivation in a dose-dependent pattern dissimilar to that of BSO-induced cellular GSH depletion. Removal of BSO allowed cells to resume proliferation. Further, growth-arresting BSO treatments were found to affect neither cell viability nor colony-forming efficiency. Addition of exogenous GSH or cysteine overcame BSO inhibition of EGF-induced DNA synthesis but not BSO depletion of cellular GSH levels. BSO was further found to inhibit the uptake of cysteine, cystine, and alpha-[1-14C]-methylaminoisobutyric acid (MeAIB) by the EGF-stimulated quiescent cells in a dose-dependent fashion. The results presented here thus demonstrate that BSO inhibits the proliferation of NRK-49F cells. This effect, however, does not correlate with BSO-induced cellular GSH depletion and is not due to an overt toxic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

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