Correlation between levels of ferritin and the iron-containing component of ribonucleotide reductase in hydroxyurea-sensitive, -resistant, and -revertant cell lines

1991 ◽  
Vol 69 (9) ◽  
pp. 635-642 ◽  
Author(s):  
Robert A. R. Hurta ◽  
Jim A. Wright
Keyword(s):  
Enzyme Activity ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Protein Concentration ◽  
Reductase Activity ◽  
M2 Protein ◽  
Wild Type ◽  
L Cells ◽  
Ribonucleotide Reductase Activity

The reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA synthesis, is catalyzed by ribonucleotide reductase. This enzyme is composed of two components, M1 and M2. Recent work has shown that inhibition of ribonucleotide reductase by the antitumor drug hydroxyurea leads to a destabilized iron centre in protein M2. We have examined the relationship between the levels of ferritin, the iron storage protein, and the iron-containing M2 component of ribonucleotide reductase. These studies were carried out with hydroxyurea-sensitive, -resistant, and -revertant cell lines. Hydroxyurea-resistant mouse L cells contained M2 gene amplification and elevated levels of enzyme activity, M2 message, and total cellular M2 protein concentration. Hydroxyurea-revertant cells exhibited a wild-type M2 gene copy number, and approximately wild-type levels of enzyme activity, M2 message, and M2 protein concentration. In addition, we observed that the hydroxyurea-resistant cells possessed elevated levels of L-chain ferritin message and total cellular H-chain ferritin protein when compared to wild-type cells. In contrast, the revertant cell population contained approximately wild-type levels of ferritin mRNA and protein. In keeping with these observations, obtained with mouse L cells, was the finding that hydroxyurea-resistant Chinese hamster ovary cells with increased ribonucleotide reductase activity exhibited elevated expression of both ferritin and M2 genes, which declined in drug-sensitive revertant hamster cell lines with decreased levels of ribonucleotide reductase activity. This is the first demonstration that reversion of hydroxyurea resistance and a decline in ribonucleotide reductase activity are accompanied by decreased ferritin expression, and supports the concept that ferritin is important in establishing resistance to hydroxyurea, and may play a role in DNA synthesis, through the regulation of functional iron-containing M2 protein levels required for ribonucleotide reduction.Key words: ribonucleotide reductase, ferritin, hydroxyurea, drug resistance.

A series of N-carbamoyloxyurea resistant cell lines with alterations in ribonucleotide reductase: lack of coordination in pyrimidine and purine reductase activity

1983 ◽  
Vol 61 (2-3) ◽  
pp. 120-129 ◽  
Author(s):  
Robert G. Hards ◽  
Jim A. Wright
Keyword(s):  
Enzyme Activity ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Reductase Activity ◽  
Resistant Cell ◽  
Activity Levels ◽  
Drug Resistant ◽  
Enzyme Substrate ◽  
Resistant Lines

N-Carbamoyloxyurea is cytotoxic for cells in culture and, like hydroxyurea and guanazole, the drug is an effective inhibitor of mammalian ribonucleotide reductase and thus DNA synthesis. In addition to ribonucleotide reductase, N-carbamoyloxyurea has a second site of action which also appears to be in the pathway of DNA synthesis. A series of drug-resistant cell lines, which contain alterations in ribonucleotide reduction, have been sequentially selected in the presence of increasing concentrations of N-carbamoyloxyurea. CDP and ADP reductase activities in these drug-resistant lines have been investigated and two types of alterations have been identified: elevated levels of enzyme activity with wild-type sensitivity to drug and altered levels of reductase with reduced drug sensitivity, probably owing to structural modification of the enzyme. Furthermore, N-carbamoyloxyurea resistant lines contain another alteration as well, presumably at a second site of drug action. They are also cross-resistant to hydroxyurea and guanazole, and studies on enzyme activity levels support our previous findings with cells selected for resistance to hydroxyurea, which showed changes in CDP reductase activity are not always coordinated with changes in ADP reductase. Although several possibilities exist, these observations are most easily explained by the existence of independent enzyme substrate binding subunits which are regulated by different mechanisms. Moreover, increases in cellular resistance were accompanied by significant increases in CDP but not ADP reductase, suggesting that an ability to maintain an adequate level of CDP reductase activity is especially important to achieve resistance to DNA synthesis inhibitors like N-carbamoyloxyurea, hydroxyurea, and guanazole.

scholarly journals Cellular adaptation to down-regulated iron transport into lymphoid leukaemic cells: effects on the expression of the gene for ribonucleotide reductase

2000 ◽  
Vol 345 (3) ◽  
pp. 681-685 ◽  
Author(s):  
Christopher R. CHITAMBAR ◽  
Janine P. WERELEY ◽  
Thomas HEIMAN ◽  
William E. ANTHOLINE ◽  
William J. O'BRIEN
Keyword(s):  
T Cells ◽  
Enzyme Activity ◽  
Ribonucleotide Reductase ◽  
Iron Transport ◽  
Reductase Activity ◽  
Wild Type ◽  
Cellular Iron ◽  
Regulatory Link ◽  
The Relationship ◽  
Ribonucleotide Reductase Activity

Ribonucleotide reductase is an iron-containing enzyme that is essential for DNA synthesis. Whereas previous studies have used various iron chelators to examine the relationship between cellular iron metabolism and ribonucleotide reductase activity in cells, they have not elucidated the relationship between iron transport into cells and the expression of the gene for ribonucleotide reductase. To investigate this, we examined ribonucleotide reductase mRNA, protein and enzyme activity in a novel line of CCRF-CEM cells (DFe-T cells) that display an approx. 60% decrease in their uptake of iron compared with the parental wild-type cell line. We found that DFe-T cells displayed an approx. 40% decrease in ribonucleotide reductase specific enzyme activity relative to wild-type cells without a change in their proliferation. Kinetic analysis of CDP reductase activity revealed an approx. 60% decrease in Vmax in DFe-T cells without a change in Km. Despite the decrease in enzyme activity, the mRNA and protein for the R1 and R2 subunits of ribonucleotide reductase in DFe-T cells were similar to those of wild-type cells. ESR spectroscopy studies revealed that DFe-T cells had a 22% decrease in the tyrosyl free radical of the R2 subunit, suggesting that a larger amount of R2 protein was present as functionally inactive apo-R2 in these cells. Our studies indicate that ribonucleotide reductase activity in CCRF-CEM cells can be down-regulated by more than 50% in response to down-regulated iron transport without an adverse effect on cell proliferation. Furthermore, our studies suggest a regulatory link between ribonucleotide reductase activity and iron transport into these cells.

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity

1990 ◽  
Vol 10 (11) ◽  
pp. 5688-5699
Author(s):  
B E Wojcik ◽  
J J Dermody ◽  
H L Ozer ◽  
B Mun ◽  
C K Mathews
Keyword(s):  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster Ovary ◽  
Reductase Activity ◽  
Chinese Hamster ◽  
Temperature Shift ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Dntp Pool ◽  
Ribonucleotide Reductase Activity

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.

Alterations in the components of ribonucleotide reductase in hydroxyurea-resistant hamster cells

1983 ◽  
Vol 3 (8) ◽  
pp. 741-748 ◽  
Author(s):  
Jim A. Wright ◽  
Joseph G. Cory
Keyword(s):  
Ribonucleotide Reductase ◽  
Reductase Activity ◽  
Antitumor Agent ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Wild Type ◽  
Type Analysis ◽  
Similar Mode ◽  
Ribonucleotide Reductase Activity ◽  
Effector Binding

Two components of mammalian ribonucleotide reductase have been separated by blue dextran-Sepharose chromatography from a hydroxyurea-resistant cell line, NcR-30A2, and its parental wild type. Analysis of reductase activity in these cells and the enzyme components reveals that there are three alterations involving ribonucleotide reductase activity in NcR-30A2 cells. There is an elevation in the effector-binding (EB) component, an elevation in the non-heine-ironcontaining (NHI) component, and an alteration in the NHI component that renders the enzyme less sensitive to inhibition by hydroxyurea. These findings easily account for the resistance of NcR-30A2 cells to the antitumor agent hydroxyurea, and to other drugs with a similar mode of action.

scholarly journals Differential accumulation of ribonucleotide reductase subunits in clam oocytes: the large subunit is stored as a polypeptide, the small subunit as untranslated mRNA.

1986 ◽  
Vol 103 (6) ◽  
pp. 2129-2136 ◽  
Author(s):  
N Standart ◽  
T Hunt ◽  
J V Ruderman
Keyword(s):  
Enzyme Activity ◽  
Early Development ◽  
Ribonucleotide Reductase ◽  
Reductase Activity ◽  
Large Subunit ◽  
Small Subunit ◽  
Maternal Mrna ◽  
Molecular Masses ◽  
Maternal Mrnas ◽  
Ribonucleotide Reductase Activity

Within minutes of fertilization of clam oocytes, translation of a set of maternal mRNAs is activated. One of the most abundant of these stored mRNAs encodes the small subunit of ribonucleotide reductase (Standart, N. M., S. J. Bray, E. L. George, T. Hunt, and J. V. Ruderman, 1985, J. Cell Biol., 100:1968-1976). Unfertilized oocytes do not contain any ribonucleotide reductase activity; such activity begins to appear shortly after fertilization. In virtually all organisms, this enzyme is composed of two dissimilar subunits with molecular masses of approximately 44 and 88 kD, both of which are required for activity. This paper reports the identification of the large subunit of clam ribonucleotide reductase isolated by dATP-Sepharose chromatography as a relatively abundant 86-kD polypeptide which is already present in oocytes, and whose level remains constant during early development. The enzyme activity of this large subunit was established in reconstitution assays using the small subunit isolated from embryos by virtue of its binding to the anti-tubulin antibody YL 1/2. Thus the two components of clam ribonucleotide reductase are differentially stored in the oocyte: the small subunit in the form of untranslated mRNA and the large subunit as protein. When fertilization triggers the activation of translation of the maternal mRNA, the newly synthesized small subunit combines with the preformed large subunit to generate active ribonucleotide reductase.

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