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Antitumor Activity and Other Biological Actions of Oligomers of Ribonuclease A

2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês

10.1074/jbc.m302711200

1083-351X

Josef Matoušek, Giovanni Gotte, P Poučková, Josef Souček, Tomáš Slavík, Francesca Vottariello, Massimo Libonati,

PARP inhibition in cancer therapy

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity. Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity. Bovine ribonuclease A oligomerizes in the forms of dimers (1Crestfield A.M. Stein W.H. Moore S. Arch. Biochem. Biophys. 1962; 1: 217-222Google Scholar), trimers, tetramers, and higher order oligomers (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar) during lyophilization from 40% acetic acid solutions. Each oligomer consists of two conformational isomers, which can be separated by cation exchange chromatography into a less basic and a more basic species (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar, 3Nenci A. Gotte G. Bertoldi M. Libonati M. Protein Sci. 2001; 10: 2017-2027Google Scholar). The molecular structures of the two dimers have been solved (4Liu Y. Hart P.J. Schlunegger M.P. Eisenberg D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3437-3442Google Scholar, 5Liu Y. Gotte G. Libonati M. Eisenberg D. Nat. Struct. Biol. 2001; 8: 211-214Google Scholar). They form by a three-dimensional domain-swapping mechanism (6Bennet M.J. Choe S. Eisenberg D. Protein Sci. 1994; 3: 1444-1463Google Scholar); the less basic dimer, formerly named minor because of its ratio of 1:4 to the more basic dimer (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar, 3Nenci A. Gotte G. Bertoldi M. Libonati M. Protein Sci. 2001; 10: 2017-2027Google Scholar, 5Liu Y. Gotte G. Libonati M. Eisenberg D. Nat. Struct. Biol. 2001; 8: 211-214Google Scholar), is formed by the swapping of the N-terminal α-helix (residues 1–15) of each monomeric subunit, and the more basic or major dimer (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar, 3Nenci A. Gotte G. Bertoldi M. Libonati M. Protein Sci. 2001; 10: 2017-2027Google Scholar, 5Liu Y. Gotte G. Libonati M. Eisenberg D. Nat. Struct. Biol. 2001; 8: 211-214Google Scholar) is formed by the swapping of the C-terminal β-strand (residues 116–124) of each monomer. On this basis, the two dimers will be called N-dimer and C-dimer, respectively. The structure of the more basic or minor trimer (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar, 3Nenci A. Gotte G. Bertoldi M. Libonati M. Protein Sci. 2001; 10: 2017-2027Google Scholar) has also been solved; it is formed by three monomers linked to each other by swapping their C-terminal β-strands, thereby forming a circular structure that looks like a propeller (7Liu Y. Gotte G. Libonati M. Eisenberg D. Protein Sci. 2002; 11: 371-380Google Scholar). It will be called the C-trimer in this paper. On the basis of its dissociation products (3Nenci A. Gotte G. Bertoldi M. Libonati M. Protein Sci. 2001; 10: 2017-2027Google Scholar, 7Liu Y. Gotte G. Libonati M. Eisenberg D. Protein Sci. 2002; 11: 371-380Google Scholar), a plausible linear model was proposed for the less basic, major trimer (its abundance is 1.5 times that of the more basic, minor trimer). In this linear model, two monomers are linked through swapping of their N termini, and a third monomer is bound to one of them by C-terminal domain swapping (5Liu Y. Gotte G. Libonati M. Eisenberg D. Nat. Struct. Biol. 2001; 8: 211-214Google Scholar, 7Liu Y. Gotte G. Libonati M. Eisenberg D. Protein Sci. 2002; 11: 371-380Google Scholar). It will be called the NC-trimer. Two linear structures for the two tetramers, the less basic minor and the more basic major (ratio, 1:1.6), have also been proposed on the basis of their dissociation products (3Nenci A. Gotte G. Bertoldi M. Libonati M. Protein Sci. 2001; 10: 2017-2027Google Scholar, 8Liu Y. Eisenberg D. Protein Sci. 2002; 11: 1285-1299Google Scholar). The first could consist of a central dimer formed by C-terminal swapping, each monomer of which is linked to another monomer by N-terminal swapping. The second model could have the opposite structure, i.e. a central dimer joined by swapped N termini connected to two more monomers by the C-terminal domain-swapping mechanism. Here, they will be called the NCN-tetramer and the CNC-tetramer, respectively. These models fit the ion exchange chromatographic behavior of the two tetramers. Moreover, two additional different structures have been proposed for the RNase A tetramers by Liu and Eisenberg (8Liu Y. Eisenberg D. Protein Sci. 2002; 11: 1285-1299Google Scholar). All RNase A oligomers, having composite active sites (His-12, His-119, and Lys-41), are enzymatically active (1Crestfield A.M. Stein W.H. Moore S. Arch. Biochem. Biophys. 1962; 1: 217-222Google Scholar, 2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar, 7Liu Y. Gotte G. Libonati M. Eisenberg D. Protein Sci. 2002; 11: 371-380Google Scholar, 9Libonati M. Bertoldi M. Sorrentino S. Biochem. J. 1996; 318: 287-290Google Scholar). Their specific activities show a 30–50% reduction on yeast RNA or poly(C) substrates compared with monomeric RNase A (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar, 9Libonati M. Bertoldi M. Sorrentino S. Biochem. J. 1996; 318: 287-290Google Scholar). Although native RNase A is inactive on double-stranded RNA (dsRNA) 1The abbreviations used are: dsRNA, double-stranded RNA; BS-RNase, bovine seminal RNase; cRI, cytosolic ribonuclease inhibitor. 1The abbreviations used are: dsRNA, double-stranded RNA; BS-RNase, bovine seminal RNase; cRI, cytosolic ribonuclease inhibitor. substrates under standard assay conditions (10Libonati M. Sorrentino S. Methods Enzymol. 2001; 341: 234-248Google Scholar), its oligomers show a remarkable depolymerizing activity on dsRNA. The extent of degradation of this RNA species increases in going from dimers to pentamers, and between each pair of same-sized oligomers, the ability to attack dsRNA is always higher for the more basic conformer (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar, 7Liu Y. Gotte G. Libonati M. Eisenberg D. Protein Sci. 2002; 11: 371-380Google Scholar, 9Libonati M. Bertoldi M. Sorrentino S. Biochem. J. 1996; 318: 287-290Google Scholar). This activity is interpreted as being due to an initial destabilization of the nucleic acid secondary structure induced by the positive charges of the RNase molecule, i.e. transient single-stranded RNA stretches would form that become susceptible to ribonuclease attack (10Libonati M. Sorrentino S. Methods Enzymol. 2001; 341: 234-248Google Scholar). A correlation has indeed been found between the number of positive charges present in the active site region of mammalian pancreatic type ribonucleases and their ability to degrade dsRNA, as well as between the basic charge "density" of the RNase A oligomers and their activity toward dsRNA (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar, 7Liu Y. Gotte G. Libonati M. Eisenberg D. Protein Sci. 2002; 11: 371-380Google Scholar, 9Libonati M. Bertoldi M. Sorrentino S. Biochem. J. 1996; 318: 287-290Google Scholar, 10Libonati M. Sorrentino S. Methods Enzymol. 2001; 341: 234-248Google Scholar). Recently, it was also shown that the efficiency of dsRNA degradation by the RNase A dimers increases under the following conditions: (i) as the distance between the active sites of the dimer decreases (which has the effect of increasing the positive charges density at the active site region); and (ii) as the orientation of the two RNA-binding patches of the oligomeric enzyme is more twisted around the molecules (7Liu Y. Gotte G. Libonati M. Eisenberg D. Protein Sci. 2002; 11: 371-380Google Scholar). These parameters also hold for the C-trimer and the bovine seminal RNase (BS-RNase) (7Liu Y. Gotte G. Libonati M. Eisenberg D. Protein Sci. 2002; 11: 371-380Google Scholar), a dimeric basic protein (pI, 10.3) having 83% sequence identity with RNase A (11Dostàl J. Matousek J. J. Reprod. Fertil. 1972; 31: 273-275Google Scholar, 12D'Alessio G. Floridi A. De Prisco R. Pignero A. Leone E. Eur. J. Biochem. 1972; 26: 153-161Google Scholar, 13D'Alessio G. Di Donato A. Parente A. Piccoli R. Trends Biochem. Sci. 1991; 16: 104-106Google Scholar), whose action toward dsRNAs is quite remarkable (10Libonati M. Sorrentino S. Methods Enzymol. 2001; 341: 234-248Google Scholar, 14Libonati M. Floridi A. Eur. J. Biochem. 1969; 8: 81-87Google Scholar). BS-RNase is also endowed with several biological actions; its aspermatogenic, embryotoxic, immunosuppressive (15Dostàl J. Matousek J. J. Reprod. Fertil. 1973; 33: 263-274Google Scholar, 16Matousek J. J. Reprod. Fertil. 1975; 43: 171-174Google Scholar, 17Soucek J. Hrubà A. Paluska E. Chudomel V. Dostàl J. Matousek J. Folia Biol. (Prague). 1983; 29: 250-261Google Scholar, 18Matousek J. Kim J.-S. Soucek J. Riha J. Ribò M. Leland P.A. Raines R.T. Comp. Biochem. Physiol. 1997; 118B: 881-888Google Scholar), and, in particular, antitumor (13D'Alessio G. Di Donato A. Parente A. Piccoli R. Trends Biochem. Sci. 1991; 16: 104-106Google Scholar, 18Matousek J. Kim J.-S. Soucek J. Riha J. Ribò M. Leland P.A. Raines R.T. Comp. Biochem. Physiol. 1997; 118B: 881-888Google Scholar, 19Matousek J. Experientia. 1973; 29: 848-850Google Scholar, 20Laccetti P. Spalletti-Cernia D. Portella G. De Corato P. D'Alessio G. Vecchio G. Cancer Res. 1994; 54: 4253-4256Google Scholar, 21D'Alessio G. Trends Cell Biol. 1993; 3: 106-109Google Scholar) activities have been extensively studied over the years. A strong antitumor action is also exerted by onconase, a ribonuclease purified from Rana pipiens oocytes (22Boix E. Wu Y. Vasandani V.M. Saxena S.K. Ardelt W. Ladner J. Youle R.J. J. Mol. Biol. 1996; 257: 992-1007Google Scholar). The lack of any significant biological activity in monomeric RNase A, and, on the contrary, the presence of various, remarkable biological actions in dimeric BS-RNase could reasonably be, at least partly, ascribed to the different quaternary structures of the two protein molecules. In fact, whereas the cytosolic ribonuclease inhibitor (cRI) (23Blackburn P. Moore S. Boyer P.D. The Enzymes. 15. Academic Press, New York1982: 317-433Google Scholar, 24Lee F.S. Vallee B.L. Prog. Nucleic Acid Res. Mol. Biol. 1993; 44: 1-30Google Scholar, 25Hofsteenge J. Curr. Opin. Struct. Biol. 1994; 4: 807-809Google Scholar) can block monomeric RNase A after its entrance into the cell, BS-RNase, because of its dimeric structure, would escape interaction with the inhibitor and therefore be able to exert its enzymatic activity in the cell (26Kim J.-S. Soucek J. Matousek J. Raines R.T. J. Biol. Chem. 1995; 270: 31097-31102Google Scholar, 27Antignani A. Naddeo M. Cubellis M.V. Russo A. D'Alessio G. Biochemistry. 2001; 40: 3492-3496Google Scholar). Based on these facts and taking into account that a significant activity against transformed cells was also shown to be displayed in vitro and in vivo by RNase A dimerized by protein engineering (28Di Donato A. Cafaro V. D'Alessio G. J. Biol. Chem. 1994; 269: 17394-17396Google Scholar), covalently cross-linked dimers and trimers of RNase A (29Bartholeyns J. Baudhuin P. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 573-576Google Scholar, 30Tarnowsky G.S. Kassel R.L. Mountain I.M. Blackburn P. Wilson G. Wang D. Cancer Res. 1976; 36: 4074-4078Google Scholar, 31Gotte G. Testolin L. Costanzo C. Sorrentino S. Armato U. Libonati M. FEBS Lett. 1997; 415: 308-312Google Scholar), and a dimeric mutant of human pancreatic RNase (32Piccoli R. Di Gaetano S. De Lorenzo C. Grauso M. Monaco C. Spalletti-Cernia D. Laccetti P. Cinatl J. Matousek J. D'Alessio G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7768-7773Google Scholar), the question arose as to whether the various RNase A oligomers obtained by the lyophilization procedure and purified as described (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar) might also be endowed with similar biological actions. We have therefore performed a series of in vitro and in vivo experiments, which demonstrate that RNase A dimers, trimers, and tetramers display aspermatogenic and antitumor activities that increase remarkably as a function of the oligomer mass and, at the same time, show a complete lack of embryotoxicity. Preparation of RNase A Oligomers—Dimers, trimers, and tetramers of RNase A (Type XII-A, purchased from Sigma) were prepared by lyophilization of the protein from 40% acetic acid solutions, as described (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar). The lyophilized material, dissolved in 0.08 m sodium phosphate buffer, pH 6.7, was subjected to ion-exchange chromatography with a Source 15S HR 16/10 or 16/50 column in a fast paced liquid chromatography (FPLC) system (Amersham Biosciences). Separation of the various RNase A species, at room temperature, was performed at pH 6.7 using a 0.085–0.18 m sodium phosphate gradient with the 16/10 column (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar) or a 0.09–0.20 m sodium phosphate gradient with the 16/50 column. The aggregates, if not used immediately, were kept diluted and frozen until use. In fact, the stability of the RNase A aggregates in sodium phosphate buffer, pH 6.7, is definitely higher at low protein concentration (3Nenci A. Gotte G. Bertoldi M. Libonati M. Protein Sci. 2001; 10: 2017-2027Google Scholar). The concentration of RNase A was estimated spectrophotometrically using ϵ2801% = 7.3 (33Wang D. Moore S. Biochemistry. 1977; 16: 2937-2942Google Scholar). Bovine Seminal RNase—Bovine seminal ribonuclease was isolated from bull seminal plasma obtained from healthy, sexually mature bulls bred in the Litohor insemination station (Litohor, Czech Republic). To isolate the enzyme, one volume of the seminal plasma was diluted with 2.5 volumes of 2% acetic acid. The protein precipitate was removed by centrifugation, and solid ammonium sulfate was added to the supernatant up to 3 m concentration. After another centrifugation, the new supernatant was then dialyzed using the Vivaflow 50 linked module flow system (Vivascience Ltd., Stonehouse, UK) and freeze-dried. Chromatographies with CM Sephadex C-50 and Sephadex G-100 columns, respectively, were used for complete purification (34Matousek J. Anim. Genet. 1994; 25: 45-50Google Scholar). Cathodic Gel Electrophoresis—Cathodic gel electrophoresis under nondenaturing conditions was performed according to Goldenberg (35Goldenberg D.P. Creighton T.E. Protein Structure. A Practical Approach. Oxford University Press, IRL Press, Oxford, UK1989: 225-250Google Scholar) with slight modifications using a β-alanine/acetic acid buffer, pH 4. Gels (12% polyacrylamide) were run at 20 mA for about 100 min at 4 °C. Fixing and staining were performed with 12.5% trichloroacetic acid and 0.1% Coomassie Brilliant Blue. Aspermatogenic Effects—Assays for the aspermatogenic action of BS-RNase or the various RNase A oligomers were carried out as described elsewhere (34Matousek J. Anim. Genet. 1994; 25: 45-50Google Scholar). A 0.01–0.05-ml volume of each sample containing 100 μg of enzyme protein was injected into the left testis of five ICR mice. After 10 days, the left and right (control) testes were excised and studied histologically. Degenerative effects were assessed, namely decreased weight of the testes, decreased width of spermatogenic layers, and reduced seminiferous tubules diameter. In Vitro Assays for Antitumor Activity—The antitumor action of the various RNase A aggregates was assayed in comparison with that of BS-RNase using two human tumor cell lines, ML-2 and HL-60, originally derived from patients suffering from acute myeloid leukemia. The cells were cultured in microtiter plates containing RPMI 1640 medium supplemented with 10% fetal calf serum exposed to a humidified atmosphere enriched with CO2 (5%, v/v). After the addition of the different enzyme preparations, the cells were cultured for 48 h. Four hours before ending the experiment, cells were pulse-labeled with 24 kBq of [6-3H]-thymidine (specific activity, 980 GBq/mmol, Institute for Research, Development and Application of Radioisotopes, Prague, Czech Republic). The biological action was expressed as counts per min (cpm), and inhibition of DNA synthesis was expressed as a percentage of controls. These experiments were carried out in triplicate. In Vivo Assays for Antitumor Activity—The antitumor action was tested in CD-1 athymic strain female outbred nude mice (AnLab Ltd., Charles River Laboratories, Prague, Czech Republic) weighing between 18 and 20 g. The mice (42Bracale A. Spalletti-Cernia D. Mastronicola M. Castaldi F. Mannucci R. Nitsch L. D'Alessio G. Biochem. J. 2002; 362: 553-560Google Scholar, divided into seven groups of six mice each) were kept under aseptic conditions in cages with bedding (SAWI-Research bedding, AnLab Ltd.) sterilized by irradiation. They were fed with a radiation-sterilized ST-1 (Bergman) diet and given autoclaved water ad libitum. Human non-pigmented melanoma (line UB900518), obtained from a surgical specimen cut in small pieces (3 × 3 mm), was transformed (stabilized) in tissue culture. This material was transplanted subcutaneously (1 × 107 cells/nude mouse, with 0.1 ml of Matrigel) into the right flank of the nude mice. Treatment was started when the area of the transplanted tumor reached the size of 5 × 5 mm (0.15–0.26 cm3), which occurred ∼14 days after inoculation. The RNase A oligomers were administered intravenously in doses of 250 μg/20 g three times a week for 4 weeks. Saline solution was administered to control animals (a group of 11 mice). Tumor sizes were measured twice a week using a slide caliper, and volume was calculated as V = a × b × π/6, where a and b are the long and short dimensions, respectively. The percentage of tumor growth inhibition (1 – (mean tumor volume in treated group/mean tumor volume in controls) × 100) was calculated and shown in Fig. 3. Embryotoxic Effects—Two-cell embryos were obtained from superovulated mice by flushing mouse oviducts ∼36 h after mating. Embryos were cultured in CZB medium (81.62 mm NaCl, 4.83 mm KCl, 1.18 mm KH2PO4, 1.18 mm MgSO4-7H2O, 25.12 mm NaHCO3, 1.7 mm CaCl2-H2O, 31.3 mm sodium lactate, 0.27 mm sodium pyruvate, 0.11 mm EDTA (disodium salt), 1 mm glutamine, 100 units/ml penicillin G sodium, 0.50 mg/ml streptomycin, and 3 mg/ml bovine serum albumin) and the protein species to be tested (μg/ml; see Table II) for 72–96 h in a humidified atmosphere containing CO2 (5% v/v) at 37.5 °C (36Chatot C.L. Ziomek C.A. Bavister B.D. Lewis J.L. Torres I. J. Reprod. Fertil. 1989; 86: 679-688Google Scholar). Controls were prepared as above, but without the protein species to be tested. The developmental stage of the embryos was monitored with a stereomicroscope.Table IIDevelopment of mice embryos after 72 hours incubation with RNase A oligomers, RNase A, or BS-RNaseEnzyme species added to the medium used for embryo cultureNumber of mice embryosNumber of embryos in cell stagesBlastocystsExpanded blastocystsTotal blastocystsBlastocysts%Control (medium)1014550RNase A913444BS-RNase1010110ND1154981CD171021270NCT1154982CT1262867NCNTT + CNCTT191321579 Open table in a new tab Aspermatogenic Activity of the RNase A Oligomers—Table I shows the results of the injection of 100 μg of each of the two conformational isomers of dimeric, trimeric, or tetrameric RNase A into the left testis of a series of mice. The parameters studied to establish the degree of the aspermatogenic effect were the weight of the testes, the width of the spermatogenic layers, and the diameter of the seminiferous tubules of testes. The action of the oligomers was compared with those of native RNase A and BS-RNase. Although the statistical significance of the data might be modest because of the small number of samples, it is sufficiently clear that, although monomeric RNase A was devoid of any effect, the activities of the RNase A oligomers were, in general, similar to those displayed by BS-RNase with the exception of the index weight of testes, which was not significantly reduced by the RNase A oligomers. Moreover, the more basic tetramer (CNC), appears to have a slightly higher aspermatogenic activity than the less basic one (NCN).Table IAspermatogenic action of RNaseA oligomers compared with that of RNase A or BS-RNaseRNase A aggregates injectedNumber of miceIndex weight of testes ± S.D.Width of spermatogenic layers of testes ± S.D.Diameter of seminiferous tubules of testes ± S.D.Degree of AspermatogensisInjected testesNon-injected testesInjected testesNon-injected testesInjected testesNon-injected testesμmμmPBS (control)641 ± 942 ± 761 ± 858 ± 10152 ± 7153 ± 130RNase A542 ± 443 ± 264 ± 362 ± 6150 ± 9156 ± 60BS-RNase619 ± 832 ± 1036 ± 362 ± 8139 ± 13165 ± 112-3ND540 ± 343 ± 330 ± 560 ± 5151 ± 11153 ± 142-3CD539 ± 340 ± 137 ± 759 ± 11151 ± 5155 ± 62-3NCT535 ± 336 ± 336 ± 463 ± 6144 ± 6157 ± 92-3CT536 ± 638 ± 128 ± 1163 ± 4143 ± 18156 ± 82-3NCNTT550 ± 1842 ± 254 ± 1260 ± 4157 ± 21155 ± 51-3CNCTT540 ± 340 ± 141 ± 1561 ± 7151 ± 22161 ± 72-3 Open table in a new tab Embryotoxic Effects of the Various Aggregates of RNase A— The development of mice embryos after 72 h incubation with the two dimers or trimers of RNase A (100 μg/ml) and a mixture of the two tetrameric conformers is shown in Table II. The action of RNase A oligomers has been tested in parallel with that of BS-RNase and native RNase A. It is quite clear that, whereas 50% of two-celled-embryos reached the blastocyst stage in control experiments and 44% in the presence of monomeric RNase A, only 10% of the embryos grew to blastocysts in the presence of BS-RNase. On the contrary, no embryotoxicity was displayed by any of the aggregated species of RNase A, in the presence of which 67–82% of the embryos formed blastocysts. In Vitro Antitumor Activity of the Oligomers of RNase A— The action of the various RNase A oligomers on ML-2 (human myeloid leukemia) cells is shown in Figs. 1, A and B and 2, A and B. The activities of the two dimeric and trimeric conformers, compared with those of BS-RNase, are shown in Fig. 1A. The two dimers and the two trimers display lower antiproliferative activities than BS-RNase. Although the N-dimer is definitely less active than the C-dimer, the two trimers show similar activities. Moreover, the C-dimer and the NC-trimer, whose charge characteristics are similar (they elute quite close to each other off cation exchange chromatography) (2Gotte G. Bertoldi M. Libonati M. Eur. J. Biochem. 1999; 265: 680-687Google Scholar), also show similar antiproliferative activities. The results obtained with the two tetramers are shown in Fig. 1B. Whereas the antiproliferative action of the NCN-tetramer is similar to or slightly lower than that of BS-RNase, the CNC-tetramer, the more basic of the two, is definitely more active than both the NCN-tetramer and BS-RNase.Fig. 2Action of RNase A oligomers and BS-RNase on the proliferation of HL-60 cells.A, action of dimers and trimers of RNase A and the action of BS-RNase on the proliferation of HL-60 cells. Experiments (in triplicate) were carried out as described in the legend to Fig. 1A. ND, N-dimer; CD, C-dimer; NCT, NC-trimer; CT, C trimer. B, action of RNase A tetramers and BS-RNase on the proliferation of HL-60 cells. Experiments (in triplicate) were carried out as described in the legend to Fig. 1A. CNCTT, CNC-tetramer; NCNTT, NCN tetramer.View Large Image Figure ViewerDownload (PPT) Qualitatively similar results were obtained by testing the antiproliferative action of RNase A dimers, trimers, or tetramers in comparison with that of BS-RNase on HL-60 cells, a human myeloid cell line. Fig. 2A shows the action of the two dimeric and trimeric conformers. With a dose of 10 μg/ml, the two dimers and the two trimers appear to display similar activities, but they are lower than the activities of BS-RNase. At doses higher than 20 μg/ml for the N-dimer and than 10 μg/ml for the other RNase A oligomers, their antiproliferative action is certainly higher than that of BS-RNase. As for the two tetramers (Fig. 2B), they are undoubtedly the most efficient antiproliferative agents; the CNC-tetramer, the more basic of the two, appears to be more active than the NCN-tetramer. In Vivo Antitumor Activity of the Oligomers of RNase A—The results of the last of three different series of experiments, which are qualitatively very similar to each other, are presented in Fig. 3. It is quite clear that all of the RNase A oligomers are active against the growth of human melanoma in nude mice with their action increasing as a function of the size of the oligomers, which is in agreement with the results obtained in the in vitro experiments performed on the ML-2 or HL-60 cell lines. The inhibition of tumor development exerted by trimers and tetramers is particularly strong, with the latter definitely being the most efficient antitumor agents. The only discrepancy concerns the reciprocal extent of action displayed by the two dimeric conformers; the more basic dimer (C-dimer) shows less activity than the less basic (N-dimer), whereas in the in vitro experiments the opposite result was found. It might also be worth mentioning that no significant changes in body weight of the six series of mice treated with dimers, trimers, or tetramers of RNase A could be noticed in the course of the experiment (data not shown). The results reported in this work show that the oligomers of bovine ribonuclease A, obtained by lyophilizing the enzyme protein from 40% acetic acid solutions, are endowed with some biological actions. As described in the Introduction, the structures of the two dimers and one of the two trimers have been determined, whereas for the second trimer and the two tetramers plausible models have been proposed on the basis of their dissociation products (3Nenci A. Gotte G. Bertoldi M. Libonati M. Protein Sci. 2001; 10: 2017-2027Google Scholar, 4Liu Y. Hart P.J. Schlunegger M.P. Eisenberg D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3437-3442Google Scholar, 5Liu Y. Gotte G. Libonati M. Eisenberg D. Nat. Struct. Biol. 2001; 8: 211-214Google Scholar, 7Liu Y. Gotte G. Libonati M. Eisenberg D. Protein Sci. 2002; 11: 371-380Google Scholar). The biological actions of the oligomers consist in an in vitro and in vivo antitumor activity (Figs. 1, 2, and 3), as well as an aspermatogenic action (Table I), similar to those ascertained for bovine seminal RNase. The RNase A oligomers lack, instead, the embryotoxic activity, which accompanies the cytotoxic action of BS-RNase (Table II). Several points need to be discussed. First, how can RNase A oligomers enter the cells? We do not have any direct evidence about this, but we might assume that they could bind to the cell surface by adsorption and then enter the cell by endocytosis, as has been suggested for BS-RNase (26Kim J.-S. Soucek J. Matousek J. Raines R.T. J. Biol. Chem. 1995; 270: 31097-31102Google Scholar, 37Soucek. J. Matousek J. Folia Biol. (Prague). 1979; 25: 142-144Google Scholar, 38Mastronicola M.R. Piccoli R. D'Alessio G. Eur. J. Biochem. 1995; 230: 242-249Google Scholar) and some RNase A variants endowed with po