Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushima-naka, Okayama 700-8530 and 1 Department of Surgery, Keio University School of Medicine,Tokyo 1608582, Japan
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Abstract |
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Keywords: cytotoxic/FGF/immunotoxin/RNase/targeting
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Introduction |
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It has been shown that upon microinjection of Xenopus oocytes, bovine RNase A inhibited protein synthesis at concentrations comparable to plant and bacterial toxins (Saxena et al., 1991). Therefore, RNases are considered to be alternative toxins because of their abilities to degrade RNA molecules. In fact, RNase A chemically conjugated to human transferrin and to antibodies to the transferrin receptor (Rybak et al., 1991
), or to human epidermal growth factor (EGF; Jinno et al., 1996b) have been shown to be cytotoxic to cells bearing the transferrin receptor or to the EGF receptor (EGFR)-overexpressing carcinoma cells, respectively. These results indicate that human enzymes belonging to the RNase A family can be used as toxins in the construction of immunotoxins (Youle et al., 1993
), although human RNases as well as RNase A are not in themselves cytotoxic. In humans, six members of the extracellular RNase A family are known. These RNases are distributed in various tissues (Rosenberg and Dyer, 1996
; Futami et al., 1997
) and possess various biological activities in addition to digestive activity (Sorrentino, 1998
). We have reported the expression of recombinant human RNase 1 which was cloned from human pancreas (Futami et al., 1995
). We have also shown that recombinant RNase 1 chemically conjugated and/or genetically fused to EGF is effective as an immunotoxin against EGFR-overexpressing human squamous carcinoma cells (Jinno et al., 1996a
; Psarras et al., 1998
). Other members of human RNases, such as RNase 2 (eosinophil-derived neurotoxin, EDN) and RNase 5 (angiogenin), have been successfully used as toxic domains of immunotoxins (Newton et al., 1994
, 1996
; Zewe et al., 1997
).
Basic fibroblast growth factor (bFGF) can regulate a number of distinct biological activities in vivo and in vitro (Basilico and Moscatelli, 1992) and is considered to be involved in the growth of a variety of neoplastic cell lines derived from melanomas (Halaban et al., 1988
), breast cancer (Penault-Llorca et al., 1995
), astronoma (Zagzag et al., 1990
) and ovarian carcinoma (Crickard et al., 1994
), and in the proliferation of mesangial cells in the glomerular disease (Floege et al., 1993
). Since overexpression of FGF receptors possibly correlate with the malignancy of tumors and bFGF is proposed to support autocrine growth of melanoma cells (Ahmed et al., 1997
), the receptors are potential targets for melanoma therapy (Kato et al., 1992
). In fact, the chemical conjugates or fusion protein between bFGF and saporin, a ribosome-inactivating protein isolated from the plant Saponaria officinalis, have been developed to show the growth inhibitory effect on the cells expressing FGF receptors (Lappi et al., 1991
, 1994
; Beith et al., 1992
).
In this paper, we designed a fused protein between human RNase 1 and human bFGF as humanized immunotoxins. The fused proteins showed cell type-specific growth inhibition against malignant cells bearing FGF receptors; however, the effect was cytostatic rather than cytotoxic.
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Materials and methods |
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Recombinant human RNase 1, des.1-7RNase 1 and human bFGF [147 amino acid form] were purified from Escherichia coli as described previously (Futami et al., 1995; Iwane et al., 1987
). Single-stranded polyribonucleotide, poly(C), a preferable substrate for human RNase 1, was purchased from Seikagaku Kogyo (Japan).
Cell culture
Mouse metastatic melanoma B16/BL6 cells (Poste et al., 1980) were grown in RPMI 1640 (Nissui, Japan) medium supplemented with 10% fetal bovine serum (FBS). Mouse BALB/c 3T3 A31 cells (Sasada et al., 1988
) were grown in Dulbecco's modified Eagle's medium (DMEM; Nissui, Japan) supplemented with 10% calf serum (CS). All media contained 70 µg/ml kanamycin as antibiotic and each cell line was maintained in a humidified atmosphere consisting of 5% CO2 in air at 37°C.
Plasmid construction of RNaseFGF fused proteins
Human RNase 1 or des.1-7RNase 1 cDNA (Seno et al., 1994; Futami et al., 1995
) was ligated to the 5'-end of the cDNA encoding human bFGF (Figure 1B
). Briefly, HindIII restriction sites were introduced into the 3'-end of human RNase 1 or des.1-7RNase 1 cDNA and the 5'-end of human bFGF cDNA by site-directed mutagenesis. Two cDNAs coding for RNaseFGF fused proteins were inserted into the T7 expression vector pET-3a (Studier and Moffat, 1986
).
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For the expression of the two fused proteins, RNase 1 and bFGF (RNF) and des.1-7RNase 1 and bFGF (des.1-7RNF), E.coli BL21(DE3)pLysS (Studier and Moffat, 1986) and E.coli MM294(DE3)pLysS (Watanabe et al., 1990
) were transformed with the plasmids described above. Each transformant was grown in LB medium containing ampicillin (50 µg/ml) and chloramphenicol (10 µg/ml) overnight at 37°C. The culture was then diluted 50-fold with LB medium and further incubated at 37°C in an incubator shaker. When an A600 of 0.6 was reached, isopropyl 1-thio-ß-D-galactopyranoside (IPTG) was added to a final concentration of 0.4 mM, and the growth was continued for a further 3 h at 37°C. Cells were harvested by centrifugation and washed once with physiological salt solution, and stored at 80°C until they were used.
Frozen cells obtained from a 1 l culture were thawed at 37°C and suspended in 100 ml lysis buffer (10 mM TrisHCl, pH 7.5, containing 10% sucrose and 0.2 M NaCl). The suspension was then homogenized (LK-21; Yamato, Japan) followed by freezethaw for complete lysis. The insoluble fraction was collected by centrifugation, resuspended in 10 ml lysis buffer, sonicated on ice for 3 min, and then centrifuged at 10 000 g for 10 min. The pellet was washed with 50 ml of 0.5% Triton X-100 containing 1 mM EDTA and 0.2 M NaCl, and then centrifuged at 10 000 g for 10 min. The resulting pellet was dissolved in 10 ml of 0.1 M TrisHCl, pH 8.5, containing 6 M guanidiniumHCl (GdmCl), and then the fused proteins were reduced with 0.1 M 2-mercaptoethanol at 37°C for 90 min under a nitrogen atmosphere. The solution was diluted to a final protein concentration of 100 µg/ml into redox buffer [10 mM TrisHCl, pH 8.5, containing 0.5 mM oxidized glutathione (Kojin, Japan), 3 mM reduced glutathione/2-mercaptoethanol, 30% (v/v) glycerol and 0.4 M GdmCl] and then incubated for 12 h at 4°C. After removal of insoluble material by centrifugation, the supernatant was applied to a heparinCellulofine (Chisso, Japan) affinity column (150x560 mm) equilibrated with buffer A [30 mM TrisHCl, pH 7.5, containing 1 mM dithiothreitol (DTT)]. The column was washed with buffer A containing 1.0 M NaCl, and the adsorbed protein was eluted with buffer A containing 2.0 M NaCl. The eluted fractions were pooled and stored at 80°C until use. Usually, the protein solution thus obtained was appropriately diluted and used for biological assay. If necessary, fused proteins were desalted by C4 reverse phase HPLC (Ultron 300 C4, 45x150 mm, Chromatopacking Center, Japan) before analysis.
Amino acid compositions of fused proteins were determined by Hitachi L8800 amino acid analyzer after hydrolysis of purified samples, and N-terminal sequences of fused proteins were determined with an Applied Biosystems ProciseTM 491 protein sequencer.
Assay of RNase activity
RNase activity using poly(C) was measured by a spectrophotomeric procedure (Sorrentino and Libonati, 1994) with some modification. Briefly, 495 µl substrate solution [0.1 M HEPESNaOH, pH 7.5, containing 0.1 M NaCl, 0.01% bovine serum albumin and 0.1 mM (in phosphodiester groups) poly(C)] was pre-incubated at 25°C. The reaction was started by adding 5 µl of the appropriate amount of each RNase or RNaseFGF fused protein and the increase in absorbance at 260 nm due to the transphosphorylation reaction catalyzed by RNase was continuously monitored with a spectrophotometer (Hitachi U-2000, Japan) at 25°C.
Heparin affinity HPLC
Purified protein solution was appropriately diluted with buffer A (described above) to reduce the NaCl concentration to 0.2 M, then applied to the heparin affinity HPLC column (Shodex AF pak HR-894, 8x50mm, Showa-denko, Japan). The elution was performed using a linear gradient of NaCl concentration from 0 to 2 M in 10 mM TrisHCl, pH 7.5, for 60 min at a flow rate of 0.5 ml/min.
Induction of tyrosine phosphorylation
Tyrosine phosphorylation induced by RNaseFGF fused proteins were detected by an anti-phosphotyrosine antibody. Briefly, equal numbers of mouse BALB/c 3T3 A31 cells (Sasada et al., 1988) were plated onto 60-mm dishes and when confluence was reached, the cells were washed and starved in DMEM containing 0.2% CS for 2 days, and then a 1 nM concentration of bFGF, RNF, des.1-7RNF, RNase 1 or 10% CS was added. After 5 min, cells were washed once with ice-cold 20 mM HEPESNaOH, pH 7.4, containing 150 mM NaCl, and harvested into 50 µl SDS-sample buffer. Samples were boiled for 3 min and 30 µl of the samples were electrophoresed on a 7.5% polyacrylamide gel, and then the proteins were transferred onto a nitrocellulose filter (0.45 µm; ADVANTEC, Japan). The blot was then blocked with 1% bovine serum albumin in TBS-T (10 mM TrisHCl, pH 7.5, containing 150 mM NaCl and 0.05% Tween 20) at 4°C, and then incubated with the primary antibody (mouse monoclonal anti-phosphotyrosine antibody clone PY20; ICN, USA) diluted to 1:1000 in TBS-T for 1 h at room temperature. After three 20-min washes with TBS-T, the secondary antibody (goat anti-mouse IgG conjugated to alkaline phosphatase; Promega, USA) diluted to 1:7500 in TBS-T was added and incubated for 1 h. The blot was then washed thoroughly and the purple color was developed in the presence of 165 µg/ml 5-bromo-4-chloro-3-indolyl-phospate and 330 µg/ml nitroblue tetrazolium (Promega, USA) in 100 mM TrisHCl, pH 9.5, 100 mM NaCl and 5 mM MgCl2.
Assays for growth inhibitory effect of RNaseFGF fused protein
The cell numbers were estimated with 3-(4,5-dimethyl thiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) as described previously (Tada et al., 1986). Exponentially growing mouse metastatic melanoma B16/BL6 cells (Poste et al., 1980
) were seeded into 96-well plates (500 cells/well; Falcon, USA) in RPMI 1640 medium supplemented with 3% FCS and incubated for 12 h at 37°C, prior to addition of the reagents. The cells were then exposed to various concentrations of reagents for 2 days. The percent of cell growth was calculated in triplicate, with the cells grown without the reagents as the control. The effects of RNF on several human carcinoma-derived cells were assessed by the same procedure, except for estimation of cell numbers with sulforhodamine B (Yamori et al., 1997) instead of MTT.
Cell cycle analysis
The effect of RNaseFGF on the cell cycle of B16/BL6 cells was analyzed by flow cytometry. B16/BL6 cells (1x106) were plated into 60-mm dishes in medium and treated with or without 5 µM RNF. After 24 h, cells were harvested and nuclear DNA was stained with propidium iodide as described by Noguchi (1991). The cellular fluorescence was measured with the FACStation (Becton Dickinson, UK).
Colony formation assay
The effects of RNaseFGF fused proteins on the proliferation of B16/BL6 cells were assessed by the number and sizes of colonies formed from 500 cells initially plated in a 35-mm dish. The cells are plated in RPMI 1640 medium supplemented with 3% FCS. After 5 h, each sample was added at a concentration of 2.5 µM. On day 5, the medium was replaced with the fresh one containing the same sample. On day 7, colonies were fixed with 10% formalin and stained with crystal violet.
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Results |
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Although the three-dimensional structure of human RNase 1 has not been determined, its structure is probably very similar to that of bovine RNase A, because all members of the RNase A family of proteins in which the three-dimensional structures have been determined, so far, are known to have common folds (Tilton et al., 1992; Acharya et al., 1994
; Mosimann et al., 1994
, 1996
; Terzyan et al., 1999
). Both N- and C-terminal regions are close to the active site cleft in RNase A (Fontecilla-Camps et al., 1994
). In contrast, RNase 1 is longer by four amino acid residues at the C-terminus and is only moderately less active than RNase A (Seno et al., 1994
). Furthermore, we have shown that truncation of seven amino acid residues in the N-terminal sequence of RNase 1 resulted in not only a much reduced activity, but also in a more pronounced reduction in the affinity to human placental RNase inhibitor (Futami et al., 1995
). As for the bFGF molecule, the C-terminal structure significantly contributes to its affinity for heparin (Seno et al., 1990
), while its N-terminal portion is flexible and not essential for its biological activity (Seno et al., 1989
; Moy et al., 1996
). Thus, we designed two fused proteins as immunotoxins based on human RNase 1 and human bFGF, as shown in Figure 1B
, in which RNF is a protein fused between the C-terminus (Thr128
Ser) of RNase 1 and the N-terminus (Leu9) of the 147-amino acid form of human bFGF, and des.1-7RNF is a 7-N-terminal-residue truncated RNF. Figure 1A
shows a schematic three-dimensional structure of RNF in which the full length of RNase A (Tilton et al., 1992
) and residues 9155 of bFGF (Moy et al., 1996
) are linked with an arbitrary 4-residue linker.
Production, refolding, purification and characterization of RNaseFGF fused proteins
The fused proteins, RNF and des.1-7RNF, were expressed in E.coli strains MM294(DE3)pLysS and BL21(DE3)pLysS. Both strains gave almost the same results. In the case of RNF expression (Figure 2), induction with IPTG led to the accumulation of recombinant proteins up to about 50% of total cellular protein (Figure 2
, lane 2). After lysis, the fused proteins were recovered as inclusion bodies, which were exhaustively washed to give a protein purity of about 90% (Figure 2
, lane 3).
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The amino acid composition and the N-terminal sequence of each RNF and des.1-7RNF were both confirmed to be coincident with the design. An N-terminal Met residue of des.1-7RNF was completely processed out as expected. The numbers of SH groups per molecule determined by colorimetric analysis using 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman, 1959) were found to be 4.18 for RNF. These values are consistent with the fact that the RNase domain should have no SH group and four SS groups, while the FGF domain should have four SH groups and no SS group.
Table I shows the RNase activity of the fused proteins and their parent RNases towards poly(C). Both RNF and des.1-7RNF retained activity and were 815-fold less active than RNase 1 and des.1-7RNase 1, respectively, suggesting that the RNase domains in these fused proteins are properly folded and their RNase activity decreased due to the introduction of the FGF domain at the C-terminus. Des.1-7RNF was 20-fold less active than RNF.
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The potential of the fused proteins to induce tyrosine phosphorylation of some cellular proteins including the major substrate of p92-95 (FGF receptor substrate 2; FRS2), which is specific to the stimulation of the FGF-receptor mediated Ras/MAPK signaling pathway (Kouhara et al., 1997), was assessed. RNF and des.1-7RNF induced the tyrosine phosphorylation of bFGF, indicating that the FGF domain in each fused protein takes the correct conformation to bind FGF receptors (Figure 3
).
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Effect of RNaseFGF on cell growth
A number of studies have suggested that bFGF may be an important autocrine growth factor for melanoma cells (Ahmed et al., 1997). Since the mouse metastatic melanoma B16/BL6 cell expresses both bFGF and high-affinity FGF receptor (Blanckaert et al., 1993
), we employed this cell line expressing FGF receptor at high levels as a model target of the RNaseFGF fused proteins. Both of the RNaseFGF fused proteins showed similar dose-dependent growth inhibitory effects on B16/BL6 cells in the micromolar range (Figure 4
). However, these growth inhibitory effects were not observed when cells were treated with an equimolar mixture of RNase 1 and bFGF.
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Discussion |
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We designed two RNaseFGF fusion proteins, RNF and des.1-7RNF, in which the C-terminal Ser128 of human RNase 1 or des.1-7RNase 1 was fused to the Leu9 of human bFGF (Figure 1), which were expressed in E.coli as inclusion bodies. Since the expression of RNase activity would inhibit host cell growth, formation of inclusion bodies should be advantageous. In this case, however, the folding into the active conformation becomes a crucial step. Nevertheless, we have obtained good yields of folded RNF (or des.1-7RNF) from inclusion bodies by SHSS interchange reaction in redox buffer (about 22 mg from a 1 l culture). One of the critical points to emerge was the use of a rather high concentration (0.4 M) of GdmCl in the folding. The presence of 0.4 M salt, such as NaCl, KCl, NH4Cl or ArgHCl, also gave good yields of the folded protein. At concentrations below 0.2 M, besides the increase in the formation of precipitates, several improperly folded species were obtained. The latter was judged from the lower affinity on a heparin column and from the elution profile of multiple peaks on a cation-exchange HPLC analysis (unpublished data). Because highly purified fused protein was efficiently folded into a single species in a salt concentration independent manner, an electrostatic interaction between the fused protein and the other materials in the inclusion bodies, such as nucleic acids, may interfere with the correct folding of the fused proteins (unpublished data).
Folded RNF and des.1-7RNF were purified using a one-step procedure by heparin affinity chromatography. RNF and des.1-7RNF, thus purified, retained well the physicochemical and biological properties originating from RNase, des.1-7RNase and bFGF. The RNase activity was decreased 815-fold (Table I), probably due to the interdomain steric hindrance rather than incorrect folding, because the stability of each domain against trypsin digestion was similar to those of the unfused proteins, respectively (data not shown).
Both RNF and des.1-7RNF showed dose-dependent growth inhibitory effects on mouse metastatic melanoma B16/BL6 cells, which are known to overexpress FGF receptors, while no inhibition was observed in a mixture of RNase 1 and bFGF under the conditions employed (in the presence of 3% serum) (Figure 4). Furthermore, the fused proteins did not show any effect on normal fibroblasts such as BALB/c 3T3 (data not shown). This growth inhibitory effect of RNF and des.1-7RNF was shown to be specific to the cells overexpressing FGF receptors, as expected. RNF and des.1-7RNF did not kill B16/BL6 cells but just slowed down the speed of cell growth (Figure 5
). However, the cells were not arrested at any particular stage in the cell cycle when treated with these fused proteins (Figure 6
). Similar results have been described in the cell growth suppression of onconase, a member of the pancreatic-type RNase family isolated from frog oocytes (Mikulski et al., 1992
). Therefore, a prolonged cell cycle may be a common property of RNase-mediated growth inhibition. We also examined apoptosis assays on B16/BL6 cells treated with 5 µM RNF by electrophoresis of internucleosomal DNA; however, no DNA fragmentation was observed. In this context, the RNaseFGF fused proteins prepared here were cytostatic rather than cytotoxic.
RNF also showed a growth inhibitory effect on the growth of many carcinoma-derived human cell lines (Table II). MDA-MB-231, HT-29 and MCF-7 are known to overexpress FGF receptors (Peyrat et al., 1991
; Murgue et al., 1994
), while the others have not yet been examined in this respect. We studied the relationship between GI50s by RNF and FGF receptor levels in MDA-MB-231 and MCF-7, which suggested the existence of two possible pathways for internalization of bFGF: high affinity receptor-mediated and low affinity receptor (heparin-like molecule)-mediated, also described previously (Roghani and Moscatelli, 1992
). These results again suggest that the cytostatic effect of the RNaseFGF fused proteins is specific to the cells overexpressing FGF receptors.
Basic FGF has been suggested to be involved in a number of proliferative diseases and pathogenesis (Baird et al., 1986), including angiogenesis, which is important in carcinogenesis (Folkman et al., 1989
). Expression of bFGF specific receptors (FGFR1) are also up-regulated in the mesangial proliferative stage of glomerular failure (Jyo, 1996
). All the cells pathologically associated with these disorders should be potential targets of a bFGF-based growth inhibitor. In the case of the proliferative disorders, such as glomerulonephritis, it may be important to suppress only the proliferation of cells without killing them. The cytostatic nature of RNF or des.1-7RNF may be useful for the treatment of such bFGF-induced proliferative disorders.
Some members of pancreatic-type RNase family, such as onconase and bovine seminal dimeric-RNase (BS-RNase), have cytotoxic activity against cancer cells and/or anti-HIV activity (Vescia et al., 1980; Wu et al., 1993
; Saxena et al., 1996
). Onconase is now in clinical trials for cancer therapy (Mikulski et al., 1993
). All investigated mammalian pancreatic-type RNases, except for BS-RNase, are known to be severely inhibited by a RNase inhibitor that occurs in the cytoplasm of virtually all mammalian cells (Futami et al., 1997
; Hofsteenge et al., 1998
). Both onconase and BS-RNase have also been reported to weakly interact with RNase inhibitor (Murthy and Sirdeshmukh, 1992
; Wu et al., 1993
).
Hence, the first mechanism of cytotoxicity of these RNases has been explained as follows. Cytotoxic RNases are adsorbed on the cell surface and internalized into cells. Second, they degrade cellular RNA evading cytosolic RNase inhibitor and inhibit protein synthesis as a result (Wu et al., 1993; Kim et al., 1995
). The mechanism proposed above explains why the cytotoxic potentials of the present RNaseFGF fusion proteins are not so high. RNaseFGF fused proteins are considered to be effectively internalized into the cytosol through bFGF receptors. Because the cytotoxic potency of an RNase should correlate with the remaining RNase activity in the presence of RNase inhibitor in the cytosol, and because cytosolic RNase inhibitor should severely inhibit the activity of human RNases, the cytotoxic effect of RNF becomes moderate. In this context, des.1-7RNF is an interesting molecule because des.1-7RNase shows a lower affinity than RNase 1 to RNase inhibitor (Futami et al., 1995
). However, the growth inhibitory effect of des.1-7RNF was almost identical to that of RNF (Figures 4 and 5
). Because the RNase activity of des.1-7RNF was 20-fold lower than that of RNF, the effect of decreased affinity on the RNase inhibitor may be compensated by the decreased RNase activity. Furthermore, the N-terminally truncated RNase domain may be unstable against proteolysis in the cytosol. This effect may also compensate for the effect of the decreased affinity to the RNase inhibitor. If we could internalize more RNF molecules relative to the level of cytosolic RNase inhibitor, a much stronger cytotoxicity would result. The balance between the rate of internalization through FGF receptors and the rate of proteolytic degradation may determine the concentration of RNF in the cytosol. Since the number of FGF receptors on a cell surface would limit the former rate, we could only change the latter rate by stabilization of the RNase domain against proteolysis by means of protein engineering. Thus, in order to potentiate the cytotoxicity of RNF, we need to design the RNase domain with a lower affinity to RNase inhibitor, high RNase activity and sufficient stability. We could successfully demonstrate by the RNaseFGF fused proteins as humanized immunotoxin of cell-type specific growth inhibitors.
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Acknowledgments |
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Notes |
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References |
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Received April 23, 1999; revised July 30, 1999; accepted August 9, 1999.