A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action Aurora Bracale 1, *, Francesco Castaldi 1, *, Lucio Nitsch 2 and Giuseppe D’Alessio 1 1 Dipartimento di Chimica Biologica and 2 Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘L. Califano’, Universita ` di Napoli, Italy The dimeric structure of seminal ribonuclease (BS-RNase) is maintained by noncovalent interactions and by two intersubunit disulfide bridges. Another unusual feature of this enzyme is its antitumour action, consisting in a cyto- toxic activity selective for malignant cells. This cytotoxic action is exerted when the protein reaches the cytosol of the affected cells, where it degrades ribosomal RNA, thus blocking protein synthesis and leading cells to death. The current model proposed for the mechanism of antitumour action of BS-RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor, a resistance due to the dimeric structure of the enzyme. Monomeric RNases, and monomeric derivatives of BS-RNase, are strongly bound by the inhibitor and inactive as antitumor agents. Here we report on mono- meric derivatives of BS-RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells. These monomers are produced by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides. We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhyd- ryl-disulfide interchange reactions, the native dimeric pro- tein, which is internalized as such, and displays its antitumour action. Keywords: antitumor; BS-RNase; disulfides; RNase. Seminal RNase from bovine seminal vesicles (BS-RNase) (reviewed in [1]) is a dimeric RNase in which two identical subunits are held together by noncovalent interactions and by two intersubunit disulfide bonds bridging Cys31 and Cys32 of one subunit with the corresponding Cys32¢ and Cys31¢ of the partner subunit. BS-RNase is an antitumour agent, as it is strongly and selectively cytotoxic for malignant cells in vitro and in vivo,withnoeffectson normal cells [2]. Since the early studies on the antitumor action of BS- RNase, it has been recognized that the dimeric structure of the enzyme is essential for its display of cytotoxic activity [3]. This conclusion was based on the lack of cytotoxic activity in a monomeric derivative of the protein obtained by selective, reductive cleavage of the intersubunit disul- fides followed by alkylation of the exposed sulfhydryls. Such conclusion has been subsequently confirmed through different experimental approaches [4], and explained [4–6] by the resistance of the enzyme in its dimeric state to the inhibitory action of CRI (the cytosolic RNase inhibitor). When the structure of CRI [7] and CRI complexed to RNases [8,9] were elucidated, it became clear how native, dimeric BS-RNase cannot fit into the horseshoe cavity of the inhibitor, whereas a monomeric form of the enzyme can, and is fully inhibited by CRI. Indeed, monomeric RNases lacking cytotoxic activity, such as bovine pancre- atic RNase and monomeric BS-RNase, could be engine- ered into cytotoxic agents by rendering them resistant to CRI [6,10]. In a survey of monomeric derivatives of BS-RNase, we found that some of them, although fully inhibited by CRI, were active as cytotoxic agents, and selective for malignant cells. Further investigation revealed that monomeric deri- vatives of BS-RNase are cytotoxic only when they conserve the intersubunit cystine residues, so that they can be re-converted into dimers, an event primed by cell sulfhydryls. These results indicate that the intersubunit disulfide bonds of BS-RNase have a key role in the mechanism of antitumour action of the enzyme. Materials and methods Materials Iodoacetic acid (IAA), iodoacetamide (IAM), 2-bromo- ethylamine hydrobromide, 5,5¢-dithio-bis(2-nitrobenzoic Correspondence to G. D’Alessio, Dipartimento di Chimica Biologica, Universita ` di Napoli ‘Federico II’, Via Mezzocannone 16, 80134 Napoli, Italy. Fax: + 39 081 5521217, Tel.: + 39 081 2534731, E-mail: dalessio@unina.it Abbreviations:BS-RNase,bovineseminalRNase;MCM,monomeric bis-Cys31,Cys32-S-carboxymethylated-BS-RNase; MCA, mono- meric bis-Cys31,Cys32-S-carboxyamidomethylated-BS-RNase; MAE, monomeric bis-Cys31,Cys32-S-aminoethylated-BS-RNase; MSSAE, monomeric bis-Cys31,Cys32-S-ethylamine-BS-RNase; MSSG, monomeric bis-Cys31,Cys32-S-glutathione-BS-RNase; CRI, cytosolic RNase inhibitor; PM, plasma membrane; IAM, iodoacetamide. *Note: These authors contributed equally to this work. (Received 23 January 2003, revised 5 March 2003, accepted 13 March 2003) Eur. J. Biochem. 270, 1980–1987 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03567.x acid) and alkaline phosphatase-conjugated anti-rabbit secondary Ig were purchased from Sigma. Reagents for Western blotting detection (SuperSignalÒ West Dura Chemiluminescent Substrate, and Immobilon TM -P mem- branes were purchased from Celbio, Milan, Italy). Poly- clonal antibodies against BS-RNase, obtained from rabbits as described previously [11], were used at a dilution of 1 : 1000. Fluorescein-tagged goat anti-rabbit secondary Ig was obtained from Jackson ImmunoResearch (West Grove, PA, USA). BS-RNase and its monomeric derivatives were prepared as described [12]. All monomers were homogene- ous upon SDS/PAGE, and catalytically active by RNase assay [13]. Other methods The monomeric derivative MSSAE (monomeric bis- Cys31,Cys32-S-ethylamine-BS-RNase, 100 lg) was labelled with 1 mCi carrier-free Na 125 I (Amersham) using IODO- BEADS (Pierce) following the manufacturer’s instructions, and desalted on PD10 Sephadex G-25 M columns (Phar- macia), equilibrated with NaCl/P i . The specific activity of labelled MSSAE was approximately 1 lCi per mg of protein. Sulfhydryl content was determined as described by [14]. RNase inhibition by the cytosolic RNase inhibitor was determined as described previously [15]. Cell cultures SV40-transformed mouse fibroblasts and the parental nontransformed Balb/C 3T3-line were obtained from American Type Culture Collection (USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-Life Technology) supplemented with 10% fetal bovine serum (Gibco-Life Technology) and Penicillin-Streptomycin-Glu- tamine 1X (Gibco-Life Technology). Cell lines were main- tained at 37 °C in a humidified incubator containing 10% CO 2 mixedwithair. Cytotoxicity assay Cells were seeded in 24- or 96-well plates (1.2 · 10 4 cellsÆ cm )2 ) in the presence of the RNase to be tested. After a 48-h incubation, cells were trypsinized, resuspended in growth medium, mixed with trypan blue solution (Sigma) (1 : 1, v/v), and counted. Cell viability was determined in triplicate as the percentage of trypan blue-excluding cells with respect to the total cell count. Preparation of cell lysates Cells treated with the RNase under test were washed first with 1 M Hepes pH 7.5 containing 0.1 M NaCl (Hepes/NaCl buffer) for 5 min, then three times with NaCl/P i , scraped from plates with a rubber policeman, collected by centrifu- gation at 1000 g, and resuspended in lysis buffer (1% NP-40 in 50 m M Tris/HCl at pH 8.0) in the presence of a protease inhibitors cocktail (CØMPLETE TM , Roche). Cells were lyzed by vortexing, incubated on ice for 30 min, and centrifuged at 16 000 g for 30 min. The final supernatant was assayed for protein concentration and frozen at )80 °C, or processed immediately. All steps were performed at 4 °C. Preparation of the membrane fraction Cells were grown to confluency in 150 mm plates, washed twice with NaCl/P i and scraped with a rubber policeman in homogenization buffer (10 m M Tris/HCl, pH 7.5, 0.25 M sucrose containing the protease inhibitors cocktail). Cells were homogenized by 25 strokes with the tight pestle of a Dounce homogenizer. The homogenate was centrifuged at 1000 g for 10 min and the supernatant was centrifuged at 16 000 g for 30 min. The pellet, representing the plasma membrane enriched fraction (PM), was resuspended in NaCl/P i , assayed for protein concentration and frozen at )80 °C or processed immediately. All steps were performed at 4 °C. Immunofluorescence studies Immunofluorescence experiments were performed as previ- ously described [11]. Briefly, mouse fibroblasts were incu- bated with the RNase under test and fixed with 3.7% formaldehyde in NaCl/P i for 15 min at room temperature. RNases were detected with the BS-RNase antiserum. To test the immunofluorescence of internalized proteins, cells were washed with Hepes/NaCl for 5 min and permeabilized with 0.1% Triton X-100 in NaCl/P i for 5 min at room temperature. Fluorescein-conjugated secondary antibody was used at a dilution of 1 : 50. Cells were visualized by epifluorescence using an Axiophot microscope (Zeiss). Results and discussion The monomeric derivatives of BS-RNase employed in this study, illustrated in Table 1, were prepared following established procedures [12] for the derivatization of Cys31 and Cys32, the cysteine residues that form the intersub- unit disulfides of BS-RNase. Briefly, MCM (monomeric bis-Cys31,Cys32-S-carboxymethylated-BS-RNase), MCA (monomeric bis-Cys31,Cys32-S-carboxyamidomethylated- BS-RNase), and MAE (monomeric bis-Cys31,Cys32-S- aminoethylated-BS-RNase) were obtained by selective reduction of the protein intersubunit disulfides followed by alkylation of the exposed sulfhydryls with iodoacetate, iodoacetamide or 2-bromoethylamine hydrobromide, respectively. The MSSAE monomer was obtained by reaction with methyl aminoethanethiosulfonate of the sulfhydryls exposed by selective reduction of the Table 1. Monomeric derivatives of BS-RNase. LC 50 is the protein concentration producing 50% of cell death. Mixed disulfide LC 50 (lgÆmL )1 ) M–(CH 2 –S–CH 2 –COO – ) 2 (MCM) No >200 M–(CH 2 –S–CH 2 –CONH 2 ) 2 (MCA) No >200 M–(CH 2 –S–CH 2 –CH 2 –NH 3 + ) 2 (MAE) No >200 M–(CH 2 –S–S–CH 2 –CH 2 –NH 3 + ) 2 (MSSAE) Yes 47 ± 6 M–(CH 2 –S–S–CH 2 –cGlu – ) 2 (MSSG) Yes 31 | Gly M–(CH 2 –S–S–CH 2 ) 2 –M (BS RNase) 25 ± 4 Ó FEBS 2003 Disulfides and antitumor action of BS-RNase (Eur. J. Biochem. 270) 1981 intersubunit disulfides. The MSSG monomer (mono- meric bis-Cys31,Cys32-S-glutathione-BS-RNase) was a by- product of the preparation of recombinant BS-RNase, in which Cys31 and Cys32 residues form mixed disulfides with glutathione moieties. All monomeric derivatives retained full RNase activity, in fact they were more active than the parent dimeric enzyme, as previously reported [12]. As for their sensitivity to the inhibitory action of the cytosolic RNase inhibitor (CRI), it is known that MCM is fully inhibited by CRI [16]. We tested MCA and MAE with increasing concentrations of CRI and found that they were inhibited by approximately 90% with a 2–4 molar excess of CRI. Monomers MSSAE and MSSG could not be tested as such for inhibition by CRI, because the strongly reducing conditions of the assay produce the cleavage of their mixed disulfides. This in turn generates, from either MSSAE or MSSG, M(SH) 2 mono- mers, i.e. BS-RNase monomers with exposed sulfhydryls at Cys31 and Cys32, and free thioethylamine or glutathione, respectively. As M(SH) 2 has been shown to be fully inhibited by CRI [5], all monomers investigated in the present study can be considered as highly sensitive to the inhibitory action of CRI. We tested the cytotoxic activity of the monomeric derivatives described above on malignant SVT2-3T3 fibro- blasts by measuring cell survival after 48 h of growth in the presence of increasing concentrations of each monomeric derivative. The data illustrated in Fig. 1 show that some monomers (MCM, MCA, MAE) have no cytotoxic activity on malignant SVT2 cells, whereas others (MSSG and MSSAE) are surprisingly cytotoxic. This cytotoxic action was selective for malignant cells, as when the latter, active monomers were tested on nonmalignant 3T3 fibroblasts, they were found to be as devoid of toxicity as native, dimeric BS-RNase (data not shown). It is noteworthy that in the inactive MCM, MCA and MAE monomers Cys31 and Cys32, the cysteine residues originally involved in the intersubunit disulfide bonding of BS-RNase, are irreversibly blocked through S-alkylation. In the active MSSAE and MSSG monomers, instead, the two Cys residues still form (mixed) disulfide bonds with thioethylamine or glutathione moieties, respectively (Table 1). This led us to hypothesize that the cytotoxic activity of the latter monomers was due to the presence in these proteins of disulfide bonds, with their potential chemical instability. It is well known that in the presence of thiolates, disulfides can undergo sulfhydryl-disulfide interchange reactions. Thus, at difference with the mono- mers bearing stable, S-alkylated Cys residues, MSSAE and MSSG monomers could, when delivered to growing cells, undergo reactions with cell thiolates, which could lead to their transformation into dimers, as described below: where M is a BS-RNase monomer, R is the thioethylamine or the glutathione moiety, CELL-S – are cell thiolates present in n molar excess, and M-(S-S) 2 -M is a reconsti- tuted dimer, in fact indistinguishable from native BS-RNase. The presence of sulphydryls on the surface of SVT2 cells was tested with 5,5¢-dithio-bis(2-nitrobenzoic acid) a reagent impermeable to cell membrane [17]. We found 63 nmol of reactive, surface sulphydryls per 10 6 SVT2 cells. In a typical experiment, this would give a molar excess of cell thiol groups of approximately 50-fold over the disulfides introduced in the cell culture upon treatment with the RNase monomers. It should be added that the intersubunit disulfides of BS-RNase are hyper-reactive to reduction, even to mild reducing agents, with respect to intrachain disulfides [18,19], and are completely cleaved by a 10-fold molar excess of dithiothreitol [19]. This hyper- reactivity is a feature also of the mixed disulfides formed by Cys31 and Cys32 with glutathione [12], and of the mixed disulfides of MSSAE (unpublished results). To verify the hypothesis described above, SVT2 fibro- blasts were grown at 37 °C in the presence of 20 lgÆmL )1 of radioactively labelled 125 I-labelled MSSAE in binding buffer (DMEM containing 1 mgÆmL )1 BSA and 25 m M Hepes at pH 7.5). At increasing time intervals, cells were washed repeatedly with NaCl/P i andthentreatedwith0.6 M NaCl in NaCl/P i for 5 min at 4 °C to detach labelled monomers bound to the cell surface [20]. The detached labelled protein was then analyzed by SDS/PAGE followed by autoradio- graphy. The results shown in Fig. 2 indicate that MSSAE monomers upon binding to the cell surface associate into a dimeric protein, with the molecular size of BS-RNase. A quantitation of the dimeric bands identified on the gel (Fig. 2) shows that MSSAE undergoes dimerization into native-like BS-RNase in a time-dependent manner, and is Fig. 1. Dose–response effects on BALB/C 3T3-SVT2 cells of mono- meric derivatives of BS-RNase. Cells were treated for 48 h at 37 °C with MCA (h), MCM (j), MAE (e), MSSAE (r), MSSG (m)or BS-RNase as a positive control (d). 2M-ðS-S-RÞ 2 þ n CELL-S À ! M-ðS-S-CELLÞ 2 þ M-ðS À Þ 2 þ 4RS À þ n-2 CELL-S À ð1Þ M-ðS-S-CELLÞ 2 þ M-ðS À Þ 2 þ n-2CELL-S À ! M-ðS-SÞ 2 -M þ n CELL-S À ð2Þ 1982 A. Bracale et al.(Eur. J. Biochem. 270) Ó FEBS 2003 almost totally dimeric after a 24-h contact with growing cells. When 125 I-labelled MSSAE was incubated in binding buffer in the absence of cells no dimerization occurred (data not shown). These results indicate that a monomeric derivative of BS- RNase in which disulfide bonds are conserved at Cys31 and Cys32 residues can reconstitute into native-like BS-RNase when administered to growing fibroblasts. Such a transfor- mation can be explained by sulfhydryl-disulfide interchange reactions occurring between cell sulfhydryls and the mixed disulfide bonds present in the monomeric derivative. We further investigated whether the MSSAE and MSSG monomers conserved the acquired dimeric structure upon cell internalization. This was considered a necessary condi- tion to attribute to the dimerization event a role in the antitumour action of BS-RNase, as BS-RNase monomers would be neutralized in the cytosol by the action of CRI. SVT2 cells were grown with MSSAE, MSSG, or native BS- RNase at a concentration of 50 lgÆmL )1 . After 24 h cells were washed at 4 °CwithNaCl/P i , then with 0.6 M NaCl to remove proteins from the cell surface. Washed cells were then lysed and analyzed by SDS/PAGE followed by immunoblotting with an anti-BS-RNase serum. The results of these experiments, illustrated in Fig. 3, show that inside the cells BS-RNase, MSSG and MSSAE are all present as dimers. These dimers are covalent, as when the electro- phoresis run was performed under reducing conditions, most of the dimeric proteins dissociated into monomers (Fig. 3). Identical results were obtained when cell lysis was carried out in the presence of 2 m M iodoacetamide (IAM) to block any free sulfhydryls (Fig. 3). This indicates that dimer formation through disulfide bonding did not occur as an artifact during lysis. These results led us to conclude that indeed BS-RNase monomers linked through disulfides to thioethylamine or glutathione moieties are reconstituted in the presence of growing fibroblasts into the parent dimeric protein, which is internalized as a native-like dimeric RNase. They also indicate for the first time that when BS-RNase is internal- ized by malignant cells, it maintains its dimeric structure. We have previously demonstrated by immunofluores- cence studies that BS-RNase binds to the surface of SVT2 cells and is internalized inside the cells, whilst the MCM monomer does not bind and is not internalized [11]. We repeated these experiments with the MSSAE monomer and treated exponentially growing SVT2 cells with 50 lgÆmL )1 of MSSAE for 75 min at 37 °C. When treated cells were tested with anti-BS-RNase serum MSSAE was found to bind effectively to their surface (Fig. 4A). SVT2 fibroblasts were then treated with MSSAE, then stripped of surface bound proteins with a high salt solution made up of 1 M Hepes pH 7.5 containing 0.1 M NaCl [11], and permeabilized with 0.1% Triton X-100. The results of this experiment, illustra- ted in Fig. 4B, show that BS-RNase immunoreactivity is localized inside the cells in endosome-like vesicles throughout the cytoplasm (Fig. 4B). These results are identical to those obtained under identical conditions with native BS-RNase [11]. Together with the results described above, they confirm that MSSAE monomers dimerize outside the cells, and are internalized as dimeric BS-RNase. As the dimerization event occurs outside the cells, before internalization, we investigated the role of plasma mem- branes (PM) in the transformation of MSSAE into a dimeric protein. 125 I-labelled MSSAE was incubated with isolated membranes from SVT2 fibroblasts (0.45 mgÆmL )1 of total protein) for 16 h at 37 °Cin0.2mLNaCl/P i . The membranes were either washed with 0.6 M NaCl in NaCl/P i , or washed with NaCl and then, after removal of the supernatant by centrifugation for 20 min at 16 000 g, treatedwith2m M dithiothreitol in NaCl/P i . Labelled proteins extracted from plasma membranes and membrane pellets were then analyzed by SDS/PAGE and autoradio- graphy. Figure 5 shows that after incubation with labelled MSSAE, membranes contained radioactive protein both monomeric and dimeric (lane 1). This indicates that under the conditions employed a substantial fraction of MSSAE was dimerized. In the fraction extracted from PM by the salt treatment (lane 2), most (approximately 80%) of the protein was dimeric. Clearly, monomers remained entrapped in the PM pellet, which upon electrophoresis in SDS was found to contain almost all monomeric protein (lane 3). When membranes were extracted with 0.6 M NaCl and the Fig. 2. Time-course of dimerization of the labelled monomeric derivative of BS-RNase 125 I-labelled MSSAE added to growing SVT2 cells. 125 I- labelled MSSAE was detached by high salt from SVT2 cells at increasing time intervals. In the insert, autoradiographic scans of the SDS/PAGE runs. D and M mark the electrophoretic mobilities of BS-RNase and monomeric BS-RNase, respectively. Fig. 3. Immunoblots of SVT2 cell lysates. Lysates were from cells treated for 24 h with BS-RNase (lane 1), monomeric MSSAE (lane 2), monomeric MSSG (lane 3), MSSAE from a lysate performed in the presence of 2 m M iodoacetamide (lane 4), BS-RNase from a lysate performed in the presence of 2 m M iodoacetamide (lane 5), MSSAE as in lane 2 after electrophoresis under reducing conditions (lane 6) and BS-RNase as in lane 1 after electrophoresis under reducing conditions (lane 7). Ó FEBS 2003 Disulfides and antitumor action of BS-RNase (Eur. J. Biochem. 270) 1983 membrane pellet was treated with dithiothreitol, the mem- brane entrapped monomers could be released (lane 4), albeit not completely, as some of them were still found to remain entrapped by PM (lane 5). The dimerization effect of PM on MSSAE was dependent on PM concentration. As shown in Fig. 6, at approximately 0.25 mgÆmL )1 of PM protein concentration, dimerization reached a plateau. These data indicate that the cell sulfhydryls responsible for the exchange with the protein disulfides are located in the plasma membrane. Furthermore, they show that, as proposed in the hypothesis above, the RNase monomers are linked through disulfides to the cell membrane, and are released only when additional sulfhydryl–disulfide exchange reactions occur, which eventually lead to their association into dimers. These results were confirmed when the separation of monomeric and dimeric RNase species produced by treating membranes with 125 I-labelled MSSAE was performed by gel filtration. In these experiments the role of membrane sulfhydryls in MSSAE dimerization was further verified by testing the effect on dimerization of iodoacetamide (IAM). 125 I-labelled MSSAE (20 lgÆmL )1 )wasaddedtocell membranes in the presence or the absence of 10 or 50 m M Fig. 4. Fluorescence studies of SVT2 fibroblasts treated with the MSSAE monomeric derivative of BS-RNase. Cells were treated with 50 lgÆmL )1 MSSAE for 75 min at 37 °C and fixed without permeabilization (A) or after a high-salt washing and permeabilization with Triton X-100 (B). The RNase was detected with anti-BS-RNase serum followed by incubation with fluorescein-tagged anti-rabbit secondary Ig. The bar represents 10 lm. 1984 A. Bracale et al.(Eur. J. Biochem. 270) Ó FEBS 2003 IAM and incubated for 16 h at 37 °C. The labelled protein extracted from PM by 0.6 M NaCl in NaCl/P i was gel-filtered on a Superdex-75 column. As shown in Fig. 7A, after 16 h of incubation with PM, MSSAE was found to be totally converted into dimers. When the incubation was carried out in the presence of 10 m M IAM, the product of Fig. 5. Autoradiography of SDS/PAGE runs of the labelled monomeric derivative of BS-RNase 125 I-labelled MSSAE incubated with plasma membranes (PM) from SVT2 cells. Lane 1, plasma membranes treated for 16 h with 125 I-labelled MSSAE; lane 2, labelled proteins extracted from PM with high salt; lane 3, labelled proteins still bound to extracted PM; lane 4, labelled proteins extracted from the PM pellet with 2 m M dithiothreitol; lane 5, proteins from the PM pellet after treatment with dithiothreitol. Fig. 6. Dimerization effect of PM isolated from SVT2 cells on the labelled monomeric derivative of BS-RNase 125 I-labelled MSSAE trea- ted with increasing concentrations of PM. Inset, autoradiographic scans of the SDS/PAGE runs of 125 I-labelled MSSAE detached by high salt from PM. D and M mark the electrophoretic mobilities of BS-RNase and monomeric BS-RNase, respectively. Fig. 7. Gel-filtration analysis of the labelled monomeric derivative of BS-RNase 125 I-labelled MSSAE after a 16-h incubation with isolated PM from SVT2 cells. The incubation was performed (A) in the absence of iodoacetamide (IAM), (B) in the presence of 10 m M IAM, or (C) of 50 m M IAM. D and M mark the elution volumes of BS-RNase and monomeric BS-RNase, respectively. Ó FEBS 2003 Disulfides and antitumor action of BS-RNase (Eur. J. Biochem. 270) 1985 dimerization decreased to 60% (Fig. 7B); at the higher IAM concentration (50 m M ), only 30% of dimer was produced (Fig. 7C). The data from the experiments on plasma membranes indicate that the cell sulfhydryls responsible for the inter- changes with disulfides, the reactions that reconstitute native-like BS-RNase, belong to the plasma membranes. They also show that BS-RNase monomers derived from MSSAE bind covalently through disulfide bonds to the membranes, as they can be released from the membranes as monomers only through the action of a reducing agent, such as dithiothreitol. The labelled RNase monomer, when added to PM, is released from the membranes as a dimeric protein, apparently produced by a sulfhydryl–disulfide interchange occurring on the membranes. These are exactly the events described in Eqns (1 and 2) of the hypothesis proposed above. It has been reported [21–23] that protein-disulfide iso- merase (PDI) is present and active at the plasma membrane surface of many types of cells. We thus considered the possibility that PDI had a role in the dimerization reaction of BS-RNase M(SSR) 2 monomers. However, we did not detect any effects of 1–10 m M concentrations of bacitracin (Sigma), a known inhibitor of PDI [21], on the dimerization reaction. Likewise, an anti-PDI serum (Stressgen) had no inhibitory effects on the reaction. These data suggest that PDI has no role in the reconstitution of dimeric BS-RNase from M(SSR) 2 monomers. Conclusion The results reported here reveal a new, significant event in the mechanism of cytotoxic action of BS-RNase on malignant cells. The event consists in the interactions, through sulfhydryl–disulfide interchange reactions, between surface cell sulfhydryls and the intersubunit disulfides that link the two subunits of BS-RNase. Monomeric derivatives of the protein are inactive as cytotoxic agents when they are prepared by reductive cleavage of the intersubunit disulfides and the resulting free sulfhydryls are blocked through alkylation. Monomers of BS-RNase are instead active when obtained by linking to small thiol compounds the sulfhy- dryls exposed after reductive cleavage. The latter monomers are found to reconstitute into disulfide linked dimers when they interact with malignant cells, or with isolated cell membranes, and are recovered as covalent dimers in treated cell lysates. Also native BS-RNase is found to be a covalent dimer inside the cells. These data lead us to conclude that the same interchange reactions occur when the native BS-RNase dimer binds and penetrate cells, with the protein undergoing a double transition from dimer to monomers linked to cell sulfhydryls, to covalent dimer again. Thus, the reported results provide a first clue to the mechanism by which BS-RNase is endocytosed by cells. Acknowledgement This work was financed by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Ministero dell’Universita ` e della Ricerca (Progetti di Rilevante Interesse Nazionale 2001) and Consorzio Interuniversitario Biotecnologie. Aurora Bracale was supported by a fellowship from Fondazione Italiana per la Ricerca sul Cancro (FIRC). References 1. D’Alessio, G., Di Donato, A., Mazzarella, L. & Piccoli, R. (1997) Seminal Ribonuclease: The Importance of Diversity. In Ribonucleases: Structures and Functions (Riordan, J.F. & D’Alessio, G., eds), pp. 383–423. Academic Press, New York, USA. 2. Youle, R.J. & D’Alessio, G. (1997) Antitumor RNases. In Ribo- nucleases: Structures and Functions (Riordan, J.F. & D’Alessio, G., eds), pp. 491–509. Academic Press, New York, USA. 3. Vescia, S., Tramontano, D., Augusti Tocco, G. & D’Alessio, G. (1980) Invitrostudies on selective inhibition of tumor cell growth by seminal ribonuclease. Cancer Res. 40, 3740–3744. 4. 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(2003) Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function. J. Biol. Chem. 278, 7607–7616. Ó FEBS 2003 Disulfides and antitumor action of BS-RNase (Eur. J. Biochem. 270) 1987 . A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action Aurora Bracale 1, *, Francesco Castaldi 1, *, Lucio Nitsch 2 and. proposed for the mechanism of antitumour action of BS -RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor,