[CANCER RESEARCH 54, 446X-4471. August 15, 19<M| Deficiency of Queuine, a Highly Modified Furine Base, in Transfer RNAs from Primary and Metastatic Ovarian Malignant Tumors in Women1 Wlodzimierz Baranowski,2 Guy Dirheimer, Jerzy Andrzej Jakowicki, and GérardKeith Second Clinic of Gynecological Surgery, Academy of Medicine, 8 Jaczewski Street, Lublin 20849. Poland fW. B., J. A. J.J, and Unité"Structure des Macromolécules Biologiques et Mécanismesde Reconnaissance, " Institut de Biologie Moléculaireet Cellulaire du CNRS, 15 rue RenéDescartes, Strasbourg 67084, France [G. D., G. K.] ABSTRACT queuine on their own despite its presence in their queuine family tRNAs. In mammals, queuine is provided by the intestinal flora or as The IUN As from rapidly growing tissues, particularly from neoplasia, a diet factor. Furthermore, in embryonic tissues and regenerating rat often exhibit queuine deficiency. In order to check whether different kinds liver (i.e., in rapidly growing tissues), the queuine family tRNAs are of ovarian tumors display queuine deficiencies we have analyzed tRNA partially queuine deficient (6, 9). Similar observations of queuine samples from 16 ovarian malignancies. The tRNAs from histologically normal myometrium (4 samples) and myoma (6 samples) were taken as deficiency have been made in queuine family tRNAs from placenta healthy tissue and benign tumor references. Queuine deficiency was de tissue despite the presence of a large amount of free queuine in the termined by an exchange assay using (8-'H]guanine and tRNA:guanine amniotic fluid (8, 10). It has in addition been observed that neoplastic transglycosylase from Escherìchiacoli. tissues very often exhibit significant deficiencies in the queuine con The mean values of queuine deficiencies in tRNAs were: 10.95 ±2.21 tent of queuine family tRNAs (9). In induced animal tumors the (SD) pmol \ ,,,„ingonadal and germ cell tumors (5 cases); 23.75 ±7.89 queuine deficiency could be correlated with the histological signs of pmol/A260 in primary epithelial tumors (9 cases); and 34.58 ±7.18 pinol malignancy (9), whereas similar observations could also be done in a A26V in metastatic tumors (2 cases). These values displayed statistically significant differences I/' = 0.0003, Kruskal-Wallis test). The queuine few cases of human malignancies, such as leukemias, lymphomas, and deficiencies in tRNAs significantly increased when moving from well- lung cancers (11, 12). differentiated through moderately differentiated to poorly differentiated Many different kinds of tumors, from benign to malignant through tumors, with the highest values found in poorly differentiated metastatic borderline malignant tumors can affect the ovary. The ovarian malig tumors i/' = 0.0002, Kruskal-Wallis test). Queuine deficiency determina nancies are generally of very poor prognosis due to silent growth and tion in tRNAs is proposed as a factor for clinical outcome prognosis of relative inaccessibility in the early stages of the diseases. Postopera- ovarian malignancies. tively, the basic factors for clinical outcome prognosis are the histo logical determination of the type of the tumor, the differentiation grade, and the tumor burden (13, 14). INTRODUCTION Our initial working hypothesis was that it might be possible that The high content of modified nucleosides is one of the character even in the early stages of the diseases the queuine content in tRNAs istic features of tRNAs. These modified nucleosides are posttranscrip- could already have been affected and could be determined in order to tionally synthesized either by direct modification of a major nucleo- consider it as a prognostic factor. We therefore undertook investiga side or by replacement of a major base by a modified one (editing) tions to estimate directly the extent of the queuine deficiency in (for a review see Refs. 1 and 2). The modified nucleosides at the first tRNAs from various human ovarian tumors in order to check for the position of the anticodon, the so-called "wobble position" in tRNA differences, if any, in relation to histopathological type of malignancy molecules, play an important role in fine tuning of codon-anticodon and grading of differentiation. interactions in tRNA-mediated message translation from mRNA into protein chain and as determinants or antideterminants of specificity of MATERIALS AND METHODS aminoacylation (for reviews see Refs. 1 and 3). The modifications at this position can be very simple, e.g., a single methylation, or very The tissues were collected from 1983 to 1992 during the operations in the complicated, as it is in the case of queuine, a highly modified purine Second Clinic of Gynecological Surgery of the Medical Academy, Lublin, (deazaguanine derivative), which is found in isoacceptor tRNAs for Poland. After surgery the samples were immediately immersed and kept in asparagine, aspartic acid, histidine, and tyrosine (queuine family liquid nitrogen until further processing. For routine histopathological charac tRNAs) (4, 5). Queuine is incorporated into tRNA posttranscription- terizations several probes from each tumor were fixed in formalin and embed ded in paraffin according to standard institutional procedure (Department of ally by a specific enzyme, the tRNA:guanine transglycosylase (EC Pathomorphology, Medical Academy, Lublin, Poland). 2.4.2.29), which mediates the exchange of the guanine by queuine As a healthy reference, the tRNAs from histologically normal myometrium without phosphodiester bond breakage (6). Investigations of the role were taken because no reference sample from normal ovary could be prepared of queuine in codon-anticodon interactions showed that queuine has due to the tissue heterogeneity of that organ. Myoma tRNA samples were used an essential function in the enhancement of correct translation of the as benign tumor controls. The reference tissues (myometrial and myoma codons (2). In addition it has been shown that queuine-lacking tRNA- specimens) were taken from the patients who were operated for reasons other Tyr in mammalian tissues could have amber suppressor activity (7). than ovarian cancer. No therapy other than surgery was applied to these Queuine, as a free base, is widely distributed in nature (8). How patients before specimens were taken. Consent statements for further analysis ever, mammals, as well as other animals, are not able to synthesize of collected tumors were obtained from each patient prior to surgery. The detailed clinical data concerning these patients are presented in Table 1. tRNA Preparation and Purification. tRNA was prepared by initial phe Received 2/17/94; accepted 6/16/94. nol extractions followed by selective isopropyl alcohol separation of the 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 accordance with nucleic acids according to the procedure of Sein el at. (15). Crude tRNA 18 U.S.C. Section 1734 solely lo indicale this fact. samples were further purified on DEAE-52 cellulose. Due to DNA contami 1This paper was presented at the European Society for Gynecologic and Obstetric nation, which was still present after Chromatographie purification, bulk tRNA Investigation "Growth and Differentiation in Reproductive Organs" Workshop, February was additionally treated by RNase-free DNase I (Promega, Madison, WI) for 6-12, 1994, Madonna di Campiglio, Italy. This work was supported by a grant from the 2 h at 37°Cin 40 mM Tris-HCI buffer, pH 7.9, containing 10 HIMNaCl and 6 French Ministry of Sciences and Technology and fellowships from the French Ministry of mM MgCU. After additional phenol extraction (0.5 h shaking at 4°C)and Sciences and Technology and Federation of European Biological Societies (W. B.). 2 To whom requests for reprints should be addressed. centrifugation, the water layer was collected and chromatographed on a Seph- 4468 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1994 American Association for Cancer Research. OUEUINE DEFICIENCY IN HUMAN OVARIAN MALIGNANT TUMORS Table 1 Queuine deficiencies in total tRNAs from human ovarian tumors and uterus tissues stage courseResultsWell (FIGO diameter examination (grade [3H]guanine PatientOvarian classification)1AIAIAnc1CIIIIIIIIIIIIIVIIIIIIIIIIIIMetastatic(cm)5126610101087881079512Histopalhologicaldifferentiation)Dysgerminomaof (pmol/A260)12.2113.488.0911.709.2523.7431.9612.2425.6335.2823.7523.h911.0324.0429.5039.663.493.675.527.157.425.326.166.293.447.614.4750.96Years5.011.06.02.5Data1.01.04.50.51.54.03.03.52.02.01.5Survival malignancy1. A.2.T. (W-D)Dysgerminoma (NSDfWell A.3.M. (W-D)Granulosa (NSD)Well T.4.K. (W-D)Granulosacell tumor (NSD)Well E.5.B. (W-D)Teratomacell lumor (NSD)not B.6.K. (W-D)Serousmalignisatum availableDiedDiedRDDiedDiedDiedDiedRDDiedDiedDied W.7.W. (M-D)Carcinomaadenocarcinoma J.8.W. (P-D)Serous anaplasticum H.9.M. (M-D)Serousadenocarcinoma A.10.M. (P-D)Serousadenocarcinoma Z.ll.G.M-R. (M-D)Serousadenocarcinoma W.12. (M-D)Serousadenocarcinoma H.13.P. (M-D)Serousadenocarcinoma J.14.J. (M-D)Serousadenocarcinoma I.15.M. (M-D)Carcinomaadenocarcinoma M.16.K. tumorMetastatic (MP-D)Neoplasmaendometriale K.ReferenceS. tumorTumor (P-D)MyometriumMyometriumMyometriumMyometriumMyomamesenchymale prob, angiogenes tissues'"17. R.18.L. H.19.K. M.20.J. B.'21.K. B.c22.K. cellulareLeiomyoma T.23.B. uteriLeiomyoma Z.24.O. uteriLeiomyoma M.25.N. uteriLeiomyoma M.26.N. uteriLeiomyoma A.E.W. uteripartim cellulareInserted coliYeast (S. cerevisiae)Age142042472257hi565952405453hi495338424339ÃŽ94344hi4544Clinical " NSD, no signs of disease; RD. recurrence of disease; W-D. well differentiated; M-D, moderately differentiated; MP-D. moderately to poorly differentiated; P-D, poorly differentiated. Due to heterogeneity of normal ovary no reference sample from that organ could be prepared. ( The same patient. adex G-25 column to remove the salts and the traces of phenol. With the use ing to a mean ±SD of 20.95 ±9.99 pmol/A260) as compared to of that method, the amount of total tRNA obtained varied from 60 to 400 /xg/g normal tissue tRNAs (myometrium, 4.96 ± 1.73 pmol//4260, depending on the kind of tumor.