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Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthe- sis pathway ... Hydroxyurea. (HU) is known to be an M2-specific inhibitor which.

Original Paper Journalof

Biomedical Science

J BiomedSci 1998;5:62-68

Received: January t7, 1997 Accepted: November t 1, 1997

A Simple and Sensitive Ribonucleotide Reductase Assay a Division of Hematology-Oncology, Departments of b Pediatrics, c Microbiology and d Pathology, Childrens Hospital of Los Angeles and the University of Southern California School of Medicine, e Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Los Angeles, Calif., USA

Key Words Ribonucleotide reductase Enzyme assay

Abstract Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [14C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg2+ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects. , e o e e e e e e e e * * * o ~ * o o ~ o ~

Introduction Ribonucleotide reductase (RR) is a key regulatory enzyme in DNA synthesis which catalyzes the de novo conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates [8, 29]. RR consists of two subunits, the M1 subunit being a Mr 170,000 homodimer with two different types of effector nucleotide binding sites and two substrate binding sites, and the M2 subunit a Mr 88,000 homodimer containing four nonheme iron molecules in the diiron-oxo sites [6, 28]. Hydroxyurea (HU) is known to be an M2-specific inhibitor which quenches a free radical in the M2 subunit of RR and inactivates its enzymatic activity [28]. As the RR M2 activity

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is linked closely to DNA synthesis and cell proliferation [1, 4, 10, 27], alterations in RR can have significant effects on the biological properties of cells such as tumor promotion and tumor progression [3, 5]. RR activity is maximal in S phase and RR is a rate-limiting enzyme for synthesis of DNA [1, 4, 8, 10, 27-29, 31]. Changes in enzyme activity and gene expression of the M 1 and M2 subunits have been observed under the regulation by transforming growth factor t31, tumor promoter 12-0tetradecanoylphorbol-13-acetate, or DNA repair after damage induced by chemotherapeutic agents [7, 14, 15, 19, 20]. HU-resistant RR cell lines show genomic amplification ofRR [ 13, 25]. Expression of the RR gene of herpes simplex virus has been shown to be required for virus

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growth a n d neoplastic t r a n s f o r m a t i o n [9, 16-18]. Deletion o f viral R R can alter the virulence and latency o f the virus in cell culture [2, 9, 12, 16, 18, 24]. Several approaches h a v e been used to measure R R enzymatic activity. One m e t h o d utilized an overnight descending p a p e r c h r o m a t o g r a p h y o f the incubation mixture to separate nucleotides, but this approach is rarely used. Most investigators employ thin-layer chromatography (TLC) to separate the reaction mixture, which requires preparation o f T L C plates and p o o r counting efficiency [11]. A third method, originally developed by M o o r e and Reichard [21 ], utilized a lengthy separation of the substrate and product m o n o p h o s p h a t e s on a D o w e x 50 H + cation exchange column [22] or borate anion exchange c h r o m a t o g r a p h y [26, 31 ]. This m e t h o d requires column monitoring, and the product is obtained in a large volume. T h e fourth method, which is the m o s t c o m m o n m e t h o d in current use, employs an Affigel 601 column [30] or a high resolution H P L C C18 column [32] to separate deoxycytidine and cytidine after dephosphorylation. All these m e t h o d s are tedious and expensive. T h e y require large a m o u n t s o f cells as starting materials, a n d only a few samples can be p e r f o r m e d in a day. In this report, we describe a novel m e t h o d to detect R R activity. T h e rationale is that the reaction substrate [14C]CDP is converted to [14C]dCDP by R R activity. Since the endogenous N D P kinase activity is m u c h stronger than R R activity in the extracts, the product [14C]dCDP is rapidly converted to [14C]dCTP providing the substrate for the subsequent D N A polymerase assay. R R activity can be accurately measured, for it is a ratelimiting step in this pathway.

Klenow fragment was pmvhased from New England Biolabs. DE-81 paper was purchased from Whatman. Herring sperm DNA, RNase A, HU, and other chemicals used in the assay were purchased from Sigma Chemical Co. (St. Louis, Mo.). Human prostate cancer cell lines DU145, PC3, AVAL were kindly provided by Dr. Teddy Fung (Childrens Hospital Los Angeles). The human oropharyngeal carcinoma cell lines KB and HURs, a HU-resistant KB done, have been described previously [31 ], and the human SMS-KCNR neuroblastoma cell line was also previously described [23].

RR NDP kinase DNA polymerase [14C]CDP .... ) [14C]dCDP , [14C]dCTP > [14C]DNA +dATP (filter +dGTP counting) +dTTP

DNA Polymerase Assay Using Random Primer Labelling Method Preparation of DNA-Primer Template. Herring DNA was digested by HaeIII or sonicated to small fragments with sizes ranging from 0.3 to 5 kb. Repeated phenol/chloroform extraction was followed by ethanol precipitation to obtain purified DNA (OD260/ OD280 = - 2 . 0 ) . The digested herring DNA was passed through a Sephadex G-50 spin column. The DNA concentration was measured and adjusted to 1 ~tg/gl, mixing with the 6-mer random primer at a final concentration of - 150 ng/gl. Calibration of Incorporation Efficiency of the Klenow Reaction. The labelling mixtures contained 90 n~vI Hepes (pH 6.6), I0 ~m'ff MgC12, 0.2 rm~ dNTP, and 5 units of the Kienow fragment. The boiled RR reaction mixture containing [14C]dCTP was added and incubated at room temperature for 30 rain. The labelling reaction was terminated by spotting the mixture onto a piece of 1.5-cm2 Whatman DE-81 filter paper. After they were air-dried, the filters were washed in a beaker containing 5% NazHPO4 by shaking gently on a platform rotator for 5 rain. Washing was repeated twice with 5 % Na2HPO4 for 2 rain each, rinsed with distilled water twice, 95% ethanol once, followed by drying under a heat lamp. Dried filter papers

W e used this coupling assay to measure R R activity in several cell extracts and c o m p a r e d it with other assay methods. This novel m e t h o d p r o v e d to be simple, fast, sensitive and inexpensive. In addition, an assay o f the R R activity for multiple samples can easily be p e r f o r m e d simultaneously.

Materials and Methods Materials [14C]CDP was purchased from Movarek. Nucleotides dNTP (dATP, dGTP, dTTP, dCTP), 6-mer random primer, and Sephadex G-25 (particle size 100-300 gin) were purchased from Pharmacia.

Novel Ribonucleotide Diphosphate Reductase Assay

Preparation of Cell Crude Extracts Approximately 1 x 10 7 viable cells (about 0.2 ml cell pellet) were collected, washed twice with buffer I [500 mM Hepes, pH 7.2; 2 mM dithiothreitol (DTT)], and resuspended in 2 ml of buffer I. RNase A was added to a final concentration of 0.2 rag/m1, and the cells were then homogenized. All steps were carried out in a cold room and in an ice bath. The homogenate was centrifuged at 16,000 g for 20 rain. To remove endogenous nucleotides the supernatant was applied into a Sephadex G-25 spin column, which had been preequilibrated with ice-cold buffer II (50 mM Hepes, pH 7.2; 2 mM DTT). After the loaded spin column was centrifuged at 1,500 g for 5 rain, the passthrough sample was collected and used for the RR assay. Protein concentration was measured using the kit from BioRAD based on Bradford method. In most cases, the protein concentration was 1-3 mg/ml in prepared crude extracts ( - 2 ml). Approximately 100-200 gg protein was used per RR assay. RR Reaction The RR assay mixture contained 50 rmV/Hepes, pH 7.2, 6 mM DTT, 4 mM/rig acetate, 2 mM ATP, and I00 pmol [14C]CDP (405 mCi/mmol). The reaction was initiated by adding RNA-free crude extracts and incubated at 37 ° C for 30 rain for the RR activity, and the reaction was terminated in boiling water for 5 rain. The substrate [14C]CDP was converted to [14C]dCDP by RR and subsequently to [lgC]dCTP by endogenous NDP kinase in the reaction mixture. This mixture was then centrifuged at 16,000 g for 5 rain to remove precipitates, and the clear supernatant containing [14C]dCTP was used for the DNA polymerase coupling assay.

J BiomedSci 1998;5:62-68

63

were then transferred to a vial with 3 ml Aquasol for scintillation counting. Under our assay condition, about the 50% of the dCTP made by RR can be incorporated into DNA templates. For nonRNase-treated controls, samples after polymerase reaction were boiled with 0.1 N KOH for 20 rain to remove incorporated RNA before spotting onto a piece of DE-81 filter paper. One unit of the RR activity is defined as the amount of the RR catalyzing the formation of 1 pmol of dCDP/60 rain at 37°C under the assay condition described as above.

Results

Time Course Study of RR Activity RR activity can be detected in crude extracts; however, a partial purification step is usually used to enrich the active components for the RR assay. A key feature of our new assay is the addition of RNase A during the homogenization step. The presence of RNase A in crude extracts eliminates RNA and prevents the high endogenous NDP kinase/RNA polymerase activities from consuming the [14C]CDP substrate used to couple the reaction of RR activity to DNA polymerase. We have tried to add CTP, the product of CDP for NDP kinase, to block the NDP kinase reaction to the RNA synthesis pathway. Actually, the result was opposite to what we expected, for CTP disappeared rapidly (incorporated into RNA), and contaminated dCTP from commercial CTP also interfered with the sensitivity of the DNA polymerase assay (decreased incorporation of the labelled [teC]dCTP). Based on these observations, RNase treatment appears to be the most effective approach to stop NDP kinase/RNA polymerase from competing for the [14C]CDP substrate. The treated homogenate was passed through Sephadex G-25 to remove degraded and endogenous nucleotides. This is essential to remove interferences for the subsequent DNA polymerase coupling assay. A typical RR assay is described in Materials and Methods, and a time course study is presented in figure 1. In this experiment, we used a prostate cell line (DU 145) and a neuroblastoma cell line (SMS-KCNR) for the RR assay. The reactions were initiated by the addition of RNase-free crude extracts and stopped at various reaction times. One was incubated in ice as the zero time control. Since the product of RR, [14C]dCDP, was rapidly converted to [14C]dCTP by endogenous NDP kinase, the subsequent DNA polymerase assay measured [14C]dCTP formation from samples at different time courses. In general, RR activity is linear for at least 30 min in both samples. The amount of substrate incorporation (1.0-1.3 pmol/30 rain/ 0.1 mg) is comparable to other reports of RR activity [11,

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J Biomed Sci 1998;5:62-68

Fig. 1. Detection of RR activity in cell extracts over time. RR activity was assayed using the standard reaction mixture described under Materials and Methods. [14C]CDP incorporation by the coupled RR/NDP kinase/DNA polymerase reaction. An aliquot of 120 lag of DU 145 extracts ( 0 ) or SMS-KCNR cell extracts ( • ) was used for the reactions and the formation of dCDP at different time points was indicated. Omission of DNA polymerase coupling was

used as a control(©).

21,22, 26, 30]. For a non-RNase control, the incorporated radioactive RNA can be removed by the heat alkaline treatment before spotting onto DE-81 paper as described in Materials and Methods. The RR activity decreases to 0.2-0.25 pmol/30 min/0.12 mg under the same reaction conditions. Thus, the result shows that the sensitivity of the RR activity without RNase treatment is much lower than that of our standard protocol.

Assay System for the RR Activities To characterize this novel assay system, we have measured the RR activity in different reaction conditions. A complete reaction mixture contains substrate [14C]CDP and some cofactors such as DTT, magnesium acetate, and ATP. The high [14CDP] conversion and incorporation into DNA by the HURs clone (fig. 2, lane 1) was defined as 100%. In the absence of ATP, the RR activity decreased to 14% of the total activity (fig. 2, lane 2). In the presence of 20 m M EDTA, the enzyme activity of this

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