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Effect of Adenosine Analogues on Protein Carboxyimethyltransferase

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Effect of Adenosine Analogues on Protein Carboxyimethyltransferase [CANCER RESEARCH 47, 3656-3661, July 15, 1987] Effect of Adenosine Analogues on Protein Car boxy Imethyltransferase, S-Adenosylhomocysteine Hydrolase, and Ribonucleotide Reducíase Activity in Murine Neuroblastoma Cells1 Robert F. O'Dea,2 Bernard L. Mirkin, Harry P. Hogenkamp, and Donna M. Barten Division of Clinical Pharmacology, Departments of Pediatrics, Pharmacology, and Biochemistry, University of Minnesota Health Sciences Center, Minneapolis, Minnesota 55455 ABSTRACT DZA, 2'-deoxyadenosine, and l-/3-D-arabinofuranosyladenine and the irreversible inhibitors AD, neplanocin A, and 3-deaza- The noncompetitive 5-adenosylhomocysteine (AdoHcy) hydrolase an neplanocin (3-7). AdoHcy hydrolase catalyzes the NAD+-de- tagonist adenosine dialdehyde (AD) has been shown to suppress the pendent oxidation of the 3'-hydroxyl group of AdoHcy (hydro- growth of cultured C-1300 murine neuroblastoma (MNB) cells. The lytic direction) or Ado (synthetic direction), resulting in the enzymatic sites at which AD and other nucleoside analogues exert their generation of 3'-keto-AdoHcy, 3'-ketoadenosine, and 3'-keto- cytotoxic effects have been postulated to include protein carboxyl- 4',5'-dehydroadenosine (2). Adenosine dialdehyde, generated methyltransferase (PCM), AdoHcy hydrolase, and ribonucleotide reduc íase. by periodate oxidation of adenosine, contains structural com AD (10~5 M) increased PCM activity 350% in suspensions prepared ponents (e.g., 3'-keto group) which are similar to the proposed from disrupted cells after 72 h of drug exposure; in contrast, 3-deaza- functional moieties of these enzyme-bound intermediates and adenosine ( IO-1 M) increased PCM activity 57%, whereas AdoHcy and has been reported to be a potent, irreversible inhibitor of sinefungin had no effect. When intact MNB cells were incubated with AdoHcy hydrolase activity from liver (5) and murine L929 AD for varying time periods up to 72 h and then pulse labeled with the leukemia cells (8). 5-adenosylmethionine precursor i,-|3H|-methionine, AD (10"' to 5 x 10"* Phosphorylated AD (S'-adenosine dialdehyde diphosphate) M) produced a concentration-dependent inhibition of protein car boxy I- inhibits bacterial ribonucleotide reducíase in vitro, the rate- methylation which persisted for up to 6 h. Following extended periods of AD treatment (48 to 72 h), AD (10~* to 10~5 M) produced a 250% limiting synthetic step in the production of deoxynucleotides increment in protein carboxylmethylation, similar in magnitude to that (9). However, the nonphosphorylated form of AD is a poor observed in disrupted cell preparations. This increase in carboxylmeth inhibitor of bacterial ribonucleotide reducíasein vitro (9), and ylation was observed at time-points when AdoHcy hydrolase activity it is uncertain whether AD is even phosphorylated in intact remained suppressed. The inhibitory effect of AD on AdoHcy hydrolase eukaryotic cells (6). These data suggest that AD may exert its activity was maximal within 4 h and still apparent after 72 h of incubation. cytotoxic effects on eukaryotic cells at other enzymatic sites. In contrast, AD treatment had no effect on the ribonucleotide reducíase Among these, PCM (EC 2.1.2.24) has been implicated as a activity of MNB cells. potential site at which AD and other Ado analogues exert their These data suggest that the cytotoxic effect of AD on MNB cells action (10). This enzyme catalyzes the AdoMet-dependent post- results directly from its inhibition of AdoHcy hydrolase activity and translational methylation of the carboxyl side chains of gluta- indirectly through its suppression of methyltransferase enzyme systems. The potential linkage between the observed long-term elevations in PCM myl and/or aspartyl residues of cellular proteins, resulting in activity and AD-induced cytotoxicity remains to be defined. the formation of labile protein methylesters (11). In prokar- yotes, the PCM system regulates chemotaxis by methylating and demethylating specific membrane proteins which modify INTRODUCTION flagellar rotation (12,13). A defined role for PCM in eukaryotes has not been provided; however, various reports have linked it The role of AdoMet3-dependent methyltransferase enzymes to the regulation of leukocyte (14,15) and, possibly, slime mold as potential targets for chemotherapeutic agents has been inten chemotaxis (16), cellular secretion (17,18), racemization repair sively examined in recent years (1). These investigations have of aging proteins (19), phototransduction (20), and cellular been focused on the development of competitive methyltrans growth and development (21-24). ferase inhibitors and agents that indirectly block intracellular The cytotoxic potency of AD and other nucleoside dialde- methylation reactions by inhibiting either AdoMet biosynthesis hydes has been determined in cultured C-1300 MNB cells and or AdoHcy metabolism. The latter approach logically derives shown to be dependent upon the presence of an adenine base from the defined biochemical function of AdoHcy hydrolase and an intact ribosyl dialdehyde moiety, structural elements (EC 3.3.1.1), the enzyme which catalyzes the reversible hydrol present in AD (30). In the following report, the effects of AD ysis of AdoHcy to adenosine and homocysteine (2). and other nucleoside analogues on PCM, AdoHcy hydrolase, A number of Ado analogues have been reported to inhibit and ribonucleotide reducíaseactivity were studied in disrupted AdoHcy hydrolase. They include the competitive antagonists cell preparations obtained from MNB cells after incubation with these agents. Received 10/24/86; revised 3/27/87; accepted 4/16/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in MATERIALS AND METHODS accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This investigation was supported by Grants NS-17194, National Institute of Adenosine Nucleoside and Dialdehyde Analogues Neurological and Communicable Diseases and Stroke, GM33776, National In stitute of General Medical Science, and the University of Minnesota Institutional All of the compounds investigated in this study were obtained from Award from the American Cancer Society. 1To whom requests for reprints should be addressed, at Division of Clinical readily available commercial sources (Sigma, St. Louis, MO; Calbi- Pharmacology, University of Minnesota Health Sciences Center, Mayo Box 468, ochem, San Diego, CA; or Lilly Research Labs, Indianapolis, IN), as 420 Delaware Street S.E., Minneapolis, MN 55455. gifts (3-deazaadenosine, Burroughs-Wellcome Research Lab, Research 3The abbreviations used are: AdoMet, S-adenosylmethiomne; AdoHcy, S- adenosylhomocysteine; MNB, murine neuroblastoma; DZA, 3-deazaadenosine; Triangle Park, NC), or synthesized in our laboratories, utilizing previ AD, adenosine dialdehyde; Ado, adenosine; PCM, protein carboxylmethyltrans- ously published procedures (5, 25). The analogues were categorized as ferase; ANOVA, analysis of variance. follows: 3656 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1987 American Association for Cancer Research. EFFECT OF ADENOSINE ANALOGUES IN MNB CELLS at 50 x g for 10 min at 4*C, and the pellets were resuspended in Nucleoside Analogues phosphate-buffered saline (pH 7.4). The washed cells were pelleted Sinefungin; DZA; AdoHcy; 5'-chloro, 5'-deoxyadenosine; 5'-deox- again by low-speed centrifugation, and the pellets were lysed in a yadenosine; 5'-chloro,5'-deoxy-ara-adenosine; 3',5'-dichloro, 2',3',5'- mixture consisting of 0.05% TRITON X-100 and 2 HIMphenylmeth- trídeoxyadenosine; methylfuryladenine; adenosine S'-carboxylic acid; ylsulfonyl fluoride in phosphate-buffered saline at a linai concentration and Ado. of IO7cells/ml. The cellular lysates were immediately frozen and stored at -70"C prior to further use. Proteins were measured by the method Dialdehyde Analogues of Lowry et al. (27). Enzyme assays for PCM and ribonucleotide AD; reduced adenosine dialdehyde; 5'-deoxyadenosine dialdehyde; reducíasewere performed with these disrupted cell preparations. inosine dialdehyde; guanosine dialdehyde; undine dialdehyde and cyti- Enzyme Assays dine dialdehyde. Dialdehydes were prepared by periodic acid oxidation of the parent nucleoside (5). PCM Activity. Aliquots of each thawed disrupted cell preparation were incubated for 10 min at 37'C in a reaction mixture containing 80 Other Chemicals HIM sodium acetate buffer, pH 6.0, and 5 x 10"' M 5-adenosyl-L- S-Adenosyl-L-[mefA>>/-3H]inethionine (specific activity, 14 Ci/mmol) (mcíA.v/-'HlnicthioniiK1(10 ^Ci/nmol) in the presence and absence of a was purchased from ICN Radiochemicals, Irvine, CA. L^methyPH] saturating concentration of the exogenous substrate, gelatin (2 mg/ml). methionine (specific activity, 80 Ci/mmol), and [2,8,5'-3H]adenosine The reactions were terminated by the addition of 1.0 ml of 10% (specific activity, 51 Ci/mmol) were purchased from New England trichloroacetic acid, followed by the addition of 100 ^1 of 1% bovine Nuclear Corp., Boston, MA. serum albumin as a coprecipitant. After centrifugation at 20,000 x g for 10 min, the supermit atils were removed, and the precipitates con MNB Tumor Cell Culture taining methylated proteins were dispersed in 0.3 ml of borate buffer (pH 11) containing 2.7% methanol as a carrier. The released methanol MNB tumors were grown in male A/J mice after s.c. implantation was extracted into 3 ml of a 3:2 toluenensoamyl alcohol mixture. After of 10" viable tumor
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