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A Novel Chimeric Protein Composed of Interleukin 13 and Pseudomonas Exotoxin Is Highly Cytotoxic to Human Carcinoma Cells Expressing Receptors for Interleukin 13 and Interleukin 4

1995; Elsevier BV; Volume: 270; Issue: 28 Linguagem: Inglês

10.1074/jbc.270.28.16775

1083-351X

Waldemar Debinski, Nicholas I. Obiri, Ira Pastan, Raj K. Puri,

Monoclonal and Polyclonal Antibodies Research

Chimeric proteins provide a unique opportunity to target therapeutic bacterial toxins to a subset of specific cells. We have generated a new recombinant chimeric toxin composed of human interleukin 13 (hIL13) and a Pseudomonas exotoxin A (PE) mutant, PE38QQR. The hIL13-PE38QQR chimera is highly cytotoxic to cell lines derived from several human epithelial carcinomas such as adenocarcinoma of stomach, colon, and skin. The cytotoxic action of hIL13-PE38QQR, which can only occur upon internalization of ligand-receptor complex, is blocked by an excess of hIL13 but not of hIL2. This action is not solely hIL13-specific because an excess of hIL4 also blocks the cytotoxicity of hIL13-toxin. Conversely, hIL13 blocks the cytotoxicity of a hIL4-PE38QQR chimera. Binding studies showed that hIL13 displaces competitively 125I-labeled hIL4-PE38QQR on carcinoma cells. These results indicate that IL4 and IL13 compete for a common binding site on the studied human cell lines. Despite this competition, hIL4 but not hIL13 decreased protein synthesis in malignant cells susceptible to the cytotoxicity of both hIL13- and hIL4-PE38QQR. Our results suggest that a spectrum of human carcinomas express binding sites for IL13. Furthermore, hIL13 and hIL4 compete reciprocally for a form of the receptor that is internalized upon binding a ligand. Thus, cancer cells represent an interesting model for studying receptors for these two growth factors. Finally, hIL13-PE38QQR may be a useful agent in the treatment of several malignancies. Chimeric proteins provide a unique opportunity to target therapeutic bacterial toxins to a subset of specific cells. We have generated a new recombinant chimeric toxin composed of human interleukin 13 (hIL13) and a Pseudomonas exotoxin A (PE) mutant, PE38QQR. The hIL13-PE38QQR chimera is highly cytotoxic to cell lines derived from several human epithelial carcinomas such as adenocarcinoma of stomach, colon, and skin. The cytotoxic action of hIL13-PE38QQR, which can only occur upon internalization of ligand-receptor complex, is blocked by an excess of hIL13 but not of hIL2. This action is not solely hIL13-specific because an excess of hIL4 also blocks the cytotoxicity of hIL13-toxin. Conversely, hIL13 blocks the cytotoxicity of a hIL4-PE38QQR chimera. Binding studies showed that hIL13 displaces competitively 125I-labeled hIL4-PE38QQR on carcinoma cells. These results indicate that IL4 and IL13 compete for a common binding site on the studied human cell lines. Despite this competition, hIL4 but not hIL13 decreased protein synthesis in malignant cells susceptible to the cytotoxicity of both hIL13- and hIL4-PE38QQR. Our results suggest that a spectrum of human carcinomas express binding sites for IL13. Furthermore, hIL13 and hIL4 compete reciprocally for a form of the receptor that is internalized upon binding a ligand. Thus, cancer cells represent an interesting model for studying receptors for these two growth factors. Finally, hIL13-PE38QQR may be a useful agent in the treatment of several malignancies. INTRODUCTIONRecent reports have described the isolation and cloning of lymphocyte growth factor interleukin 13 (IL13)1 1The abbreviations used are: ILinterleukinhhumanRreceptorγcγ-subunit of the interleukin 2 receptorPEPseudomonas exotoxin ArrecombinantPCRpolymerase chain reactionIC5050% inhibitory concentrationTGFtransforming growth factor. (1McKenzie A.N.J. Culpepper J.A. de Waal Malefyt R. Brière F. Punnonen J. Aversa G. Sato A. Dang W. Cocks B.G. Menon S. de Vries J.E. Banchereau J. Zurawski G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3735-3739Crossref PubMed Scopus (530) Google Scholar, 2Minty A. Chalon P. Derocq J.-M. Dumont X. Guillemot J.-C. Kaghad M. Labit C. Leplatois P. Liauzun P. Miloux B. Mointy C. Casselas P. Loison G. Lupker J. Shire D. Ferrara P. Caput D. Nature. 1993; 362: 248-250Crossref PubMed Scopus (841) Google Scholar). IL13 is a potentially glycosylated peptide of Mr 12,000, and it bears a limited but significant homology to IL4 at the N and C termini of the protein chain. Both ends of IL4 are important for binding to receptor (3, 4). Functional responses of normal blood cells to IL13 are pleiotropic and often, but not always, overlapping with those observed using IL4(5Jacobsen S.E.W. Okkenhaug C. Veiby O.P. Caput D. Ferrara P. Minty A. J. Exp. Med. 1994; 180: 75-82Crossref PubMed Scopus (48) Google Scholar, 6Sironi M. Sciacca F.L. Matteucci C. Conni M. Vecchi A. Bernasconi S. Minty A. Caput D. Ferrara P. Colotta F. Mantovani A. Blood. 1994; 84: 1913-1921Crossref PubMed Google Scholar). Contrary to IL4, IL13 is not species-specific and does not regulate T lymphocytes(1McKenzie A.N.J. Culpepper J.A. de Waal Malefyt R. Brière F. Punnonen J. Aversa G. Sato A. Dang W. Cocks B.G. Menon S. de Vries J.E. Banchereau J. Zurawski G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3735-3739Crossref PubMed Scopus (530) Google Scholar, 2Minty A. Chalon P. Derocq J.-M. Dumont X. Guillemot J.-C. Kaghad M. Labit C. Leplatois P. Liauzun P. Miloux B. Mointy C. Casselas P. Loison G. Lupker J. Shire D. Ferrara P. Caput D. Nature. 1993; 362: 248-250Crossref PubMed Scopus (841) Google Scholar). It has been suggested that receptors for hIL4 and hIL13 share a subunit that is important for ligand-induced intracellular signaling(7Zurawski S.M. Bega Jr., F. Huyghe B. Zurawski G. EMBO J. 1993; 12: 2663-2670Crossref PubMed Scopus (439) Google Scholar). This is based on observation that an antagonist of IL4, a mutated form of hIL4, blocked the mitogenic effect of both hIL4 and hIL13 on pre-leukemic TF 1 cells. As hIL13 weakly displaced labeled hIL4 from binding sites, nonmutated hIL4 competition for hIL13 sites was not reported(7Zurawski S.M. Bega Jr., F. Huyghe B. Zurawski G. EMBO J. 1993; 12: 2663-2670Crossref PubMed Scopus (439) Google Scholar). In fact, our experiments showed lack of displacement of labeled hIL13 by a wild-type hIL4 on TF 1 cells and a very weak or nil displacement of hIL13 by hIL4 on two renal cell carcinoma cell lines(8Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Also, a possibility has been raised that hIL13R may use the γc subunit of the hIL2R, as has been observed with hIL4R and hIL7R(9Kondo M. Takeshita T. Ishii N. Nakamura M. Watanabe S. Arai K.-i. Sugamura K. Science. 1993; 262: 1874-1877Crossref PubMed Scopus (733) Google Scholar, 10Noguchi M. Nakamura Y. Russell S.M. Ziegler S.F. Tsang M. Cao X. Leonard W.J. Science. 1993; 262: 1877-1880Crossref PubMed Scopus (781) Google Scholar, 11Russell S.M. Keegan A.D. Harada N. Nakamura Y. Noguchi M. Leland P. Friedmann M.C. Miyajima A. Puri R.K. Paul W.E. Leonard W.J. Science. 1993; 262: 1880-1883Crossref PubMed Scopus (740) Google Scholar).We have recently reported that a wide range of human cancer cell lines express receptors for hIL4; these include hematopoietic and epithelial malignancies(12Debinski W. Puri R.K. Kreitman R.J. Pastan I. J. Biol. Chem. 1993; 268: 14065-14070Abstract Full Text PDF PubMed Google Scholar, 13Obiri N.I. Hillman G.G. Hass G.P. Sud S. Puri R.K. J. Clin. Invest. 1993; 91: 88-93Crossref PubMed Scopus (171) Google Scholar). When the hIL4R was targeted with PE-based chimeric toxins, we observed a specific killing of cancer cells expressing hIL4R(12Debinski W. Puri R.K. Kreitman R.J. Pastan I. J. Biol. Chem. 1993; 268: 14065-14070Abstract Full Text PDF PubMed Google Scholar, 14Puri R.K. Debinski W. Obiri N. Kreitman R. Pastan I. Cell Immunol. 1994; 154: 369-379Crossref PubMed Scopus (23) Google Scholar). We have also shown that the PE-containing chimeric toxins have prominent antitumor activities in a human solid tumor xenograft model(15Debinski W. Puri R.K. Pastan I. Int. J. Cancer. 1994; 58: 744-748Crossref PubMed Scopus (22) Google Scholar).PE is a single-chain bacterial protein made up of three major domains (for review, see Ref. 16). A characteristic feature of PE is that the N-terminally located domain Ia binds to the α2-macroglobulin receptor, and the ligand-receptor complex undergoes receptor-mediated internalization (endocytosis) to allow intracellular routing and processing of the toxin(16Pastan I. Chaudhary V.K. FitzGerald D. Annu. Rev. Biochem. 1992; 61: 331-354Crossref PubMed Scopus (311) Google Scholar). Domain II is a site of proteolytic cleavage that activates PE and is necessary to catalyze the translocation of the toxin into the cytosol. The C-terminal domain, domain III, contains the REDLK sequence that directs the processed fragment of the toxin to the endoplasmic recticulum and possesses an ADP ribosylation activity that inactivates elongation factor 2 and leads to cell death(16Pastan I. Chaudhary V.K. FitzGerald D. Annu. Rev. Biochem. 1992; 61: 331-354Crossref PubMed Scopus (311) Google Scholar). Since domain Ia of PE binds to many types of eukaryotic cells, several mutated forms of the exotoxin have been constructed that do not bind to its ubiquitous receptor and can be attached at a gene level to various internalized ligands. One of the derivatives of PE used in the present study is PE38QQR in which domain Ia (amino acids 1-252) and amino acids 365-380 of PE are deleted, and lysine residues at positions 590 and 606 are replaced by glutamine and at 613 replaced by arginine(17Debinski W. Pastan I. Bioconjugate Chem. 1994; 5: 40-46Crossref PubMed Scopus (33) Google Scholar).Because hIL4R are richly represented among malignancies and because of the similarities between hIL4 and hIL13, we used human cancer cell lines to study the interaction of hIL13 and hIL4 with their receptors. We have used a novel recombinant chimeric toxin in which hIL13 is fused to PE38QQR, since fusion proteins containing PE are a powerful mean of plasma membrane receptors analysis(18Waldmann T.A. Pastan I.H. Gansow O.A. Junghans R.P. Ann. Int. Med. 1992; 116: 148-160Crossref PubMed Scopus (93) Google Scholar).EXPERIMENTAL PROCEDURESMaterialsRestriction endonucleases and DNA ligase were obtained from New England Biolabs (Beverly, MA), Life Technologies, Inc., and Boehringer Mannheim. [3H]Leucine and 125I were purchased from Amersham Corp. Fast protein liquid chromatography columns and media were purchased from Pharmacia Biotech Inc. Oligonucleotide primers were synthesized at Pharmacia's Gene Assembler at the Research Center, Hôtel-Dieu Hospital, Montreal. PCR kit was from Perkin-Elmer.Plasmids, Bacterial Strains, and Cell LinesPlasmids carry a T7 bacteriophage late promoter, a T7 transcription terminator at the end of the open reading frame of the protein, a f1 origin of replication and gene for ampicillin resistance(19Debinski W. Karlsson B. Lindholm L. Siegall C.B. Willingham M.C. FitzGerald D. Pastan I. J. Clin. Invest. 1992; 90: 405-411Crossref PubMed Scopus (39) Google Scholar). The gene encoding hIL13 (a generous gift of Dr. René de Waal Malefyt of the DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA to W.D.; plasmid JFE14-SRα) was PCR-cloned into plasmid pWDMH-QQR to produce hIL13-PE38QQR or into plasmid pSGC242FdN1(20Debinski W. Pastan I. Clin. Cancer Res. 1995; (in press)Google Scholar), to make hIL13 alone. hIL4 was cloned into an expression vector in a similar way to hIL13 using plasmid pWDMH4 (12Debinski W. Puri R.K. Kreitman R.J. Pastan I. J. Biol. Chem. 1993; 268: 14065-14070Abstract Full Text PDF PubMed Google Scholar) as a template for PCR amplification. Recombinant proteins were expressed in Escherichia coli BL21 (λDE3) under control of the T7 late promoter(12Debinski W. Puri R.K. Kreitman R.J. Pastan I. J. Biol. Chem. 1993; 268: 14065-14070Abstract Full Text PDF PubMed Google Scholar). Plasmids were amplified in E. coli (HB101 or DH5α high efficiency transformation) (Life Technologies, Inc.) and DNA was extracted using Qiagen kits (Chatsworth, CA).Plamids ConstructionThe chimeric gene encoding hIL13-PE38QQR (phuIL13-Tx) was constructed as outlined (see Fig. 1). hIL13 was PCR-amplified from plasmid JFE14-SRα in its mature form. New sites were introduced for the restriction endonucleases NdeI and HindIII at the 5′ and 3′ ends of the hIL13 gene, respectively. The 336-base pair DNA fragment was obtained during 25 cycles of PCR and digested with appropriate restriction enzymes. The digested fragment was subcloned into the vector obtained by digestion of plasmid pWDMH4-QQR (15Debinski W. Puri R.K. Pastan I. Int. J. Cancer. 1994; 58: 744-748Crossref PubMed Scopus (22) Google Scholar) with NdeI and HindIII to produce plasmid phuIL13-Tx. The 5′ end of the gene fusion was sequenced and showed the correct DNA of hIL13.Expression and Purification of Recombinant ProteinsE. coli BL21 (λDE3) cells were transformed with plasmids of interest and cultured in 1.0 liter of Terrific Broth (Life Technologies, Inc.). hIL13 and hIL13-PE38QQR were localized to the inclusion bodies. The procedure for the recombinant proteins isolation from the inclusion bodies was described previously(12Debinski W. Puri R.K. Kreitman R.J. Pastan I. J. Biol. Chem. 1993; 268: 14065-14070Abstract Full Text PDF PubMed Google Scholar). After dialysis, the renatured protein of hIL13-PE38QQR was purified on Q-Sepharose Fast Flow and by size exclusion chromatography on Sephacryl S-200 HR (Pharmacia). The initial step of hIL13 or hIL4 purification was conducted on SP-Sepharose Fast Flow (Pharmacia).Protein concentration was determined by the Bradford assay (Pierce “Plus,” Rockford, IL) using bovine serum albumin as a standard.Protein Synthesis Inhibition AssayThe cytotoxic activity of chimeric toxins, such as hIL13-PE38QQR, were tested as follows. Usually 1 × 104 cells/well were plated in a 24-well tissue culture plate in 1 ml of medium, and various concentrations of the chimeric toxins were added 20-28 h following cell plating. After 20 h of incubation with chimeric toxins, [3H]leucine was added to cells for 4 h, and the cell-associated radioactivity was measured. For blocking studies, rhIL2, −4, or −13 was added to cells for 30 min before the chimeric toxin addition. Data were obtained from the average of duplicates, and the assays were repeated several times.To evaluate the effects of cytokines on the protein synthesis, the assays were performed as follows. 2 × 103 cells/well were plated in a 24-well tissue culture plate in 1 ml of medium, and the cytokines were added 20 h following cell plating. After 72 h of incubation with the cytokines, [3H]leucine was added to the cells for 4 h, and the cell-associated radioactivity was measured.Competitive Binding AssayWe determined the binding ability of hIL13 as compared with hIL4-PE38QQR. The recombinant hIL4-PE38QQR was labeled with 125I using the lactoperoxidase method exactly as described previously(19Debinski W. Karlsson B. Lindholm L. Siegall C.B. Willingham M.C. FitzGerald D. Pastan I. J. Clin. Invest. 1992; 90: 405-411Crossref PubMed Scopus (39) Google Scholar). The specific activity of 125I-hIL4-PE38QQR was 6.2 μCi/μg of protein. Binding assays were performed by a standard saturation and displacement curves analysis. A431 epidermoid carcinoma cells were seeded at 105 cells/well in a 24-well tissue culture plates at 24 h before the experiment. The plates were placed on ice, and cells were washed with ice-cold PBS without Ca2+ or Mg2+ in 0.2% bovine serum albumin, as described previously(19Debinski W. Karlsson B. Lindholm L. Siegall C.B. Willingham M.C. FitzGerald D. Pastan I. J. Clin. Invest. 1992; 90: 405-411Crossref PubMed Scopus (39) Google Scholar). Then, increasing concentrations of hIL13 or hIL4-PE38QQR were added to cells and incubated 30 min prior to the addition of fixed amount of 125I-hIL4-PE38QQR for 2-3 h. After incubation, the cells were washed twice and lysed with 0.1 NAOH, and the radioactivity was counted in a γ counter.RESULTSTo construct the chimeric toxin, the coding region of the hIL13 gene was fused to a gene encoding a mutated form of PE, PE38QQR (Figs. 1 and 2A). The chimeric gene is in the bacterial vector under the control of a bacteriophage T7 late promoter; the protein was expressed in E. coli BL21 (λDE3) as described previously (12Debinski W. Puri R.K. Kreitman R.J. Pastan I. J. Biol. Chem. 1993; 268: 14065-14070Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). hIL13 alone was subcloned into the same expression plasmid. hIL13 and hIL13-PE38QQR were expressed at high levels in bacteria as seen in SDS-polyacrylamide gel electrophoresis analysis of the total cell extract (Fig. 2B). After initial purification on SP-Sepharose (hIL13) or Q-Sepharose (hIL13-PE38QQR), the renatured recombinant proteins were applied onto a Sephacryl S-200 HR Pharmacia column (Fig. 2B). hIL13 and hIL13-PE38QQR appeared as single entities demonstrating the very high purity of the final products. The chimeric toxin migrated within somewhat lower than expected for 50-kDa protein Mr range, which may be related to the hydrophobicity of the molecule. The biologic activity of the rhIL13 was exactly the same as one obtained from Sanofi Elf Bio Recherche(8Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar).Figure 2:A, Schematic drawing of multi-domain proteins: (i) PE, (ii) its derivative, PE38QQR, and (iii) hIL13-PE38QQR. Circles, structural domains of PE; domain Ia (amino acids 1-252), binding domain; domain II (amino acids 253-364), place of the proteolytic cleavage shown by an interrupted line; the dispensable domain Ib (amino acids 365-404); and domain III (amino acids 405-613), the ADP-ribosylating domain. PE38QQR, domain Ia and amino acids 365-380 in Ib are deleted, plus the three lysine residues in domain III at positions 590, 606, and 613 are changed to two glutamines and arginine (QQR) (17). The square symbolizes hIL13. B, expression in E. coli and purification of hIL13 and hIL13-PE38QQR. 15% nonreduced SDS-polyacrylamide gel electrophoresis stained with Coomassie Blue. Total cell extract of transformed E. coli BL21 (λDE3) was loaded at 2 μl/lane (sucrose suspension of the pellet obtained from 1.0 liter of culture; total volume was 75 ml). Sephacryl S-200 HR-purified proteins were loaded at 5.5 μg/lane of hIL13 and 11 μg/lane hIL13-PE38QQR, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)hIL13-PE38QQR Is Cytotoxic to Many Cancer Cell LinesThere have been no reports about the presence of IL13R on human solid cancers besides on renal cell carcinomas(8Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Therefore, we tested several established cancer cell lines to determine if hIL13-PE38QQR is cytotoxic to them. We examined cancers derived from colon, skin, and stomach; the cancer cells were sensitive to hIL13-PE38QQR with IC50 values ranging from less than 1 ng/ml to 300 ng/ml (20 pM to 6.0 nM). A colon adenocarcinoma cell line, Colo201, was very responsive with an IC50 of 1 ng/ml (Fig. 3). A431 epidermoid carcinoma cells were also very sensitive to the action of hIL13-toxin; the IC50 for hIL13-PE38QQR ranged from 6 to 10 ng/ml. A gastric carcinoma CRL1739 cell line responded moderately to the hIL13-toxin with an IC50 of 50 ng/ml. Another colon carcinoma cell line, Colo205, had a poorer response with an IC50 of 300 ng/ml. The cytotoxic action of hIL13-PE38QQR was specific as it was blocked by a 10-fold excess of hIL13 on all cells (Fig. 3). These data suggest that a specturm of human cancer cells possess hIL13 binding sites and such cells are sensitive to hIL13-PE38QQR chimeric toxin.Figure 3:Cytotoxic activity of hIL13-PE38QQR on several cancer cell lines and an inhibition of this cytotoxicity by hIL13. hIL13 was added at a concentration of 1.0 μg/ml. The dashed line shows 50% of [3H]leucine incorporation. ● hIL13-PE38QQR; ○, with hIL13.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because the hIL13R has been suggested to share γc subunit of the IL2R(11Russell S.M. Keegan A.D. Harada N. Nakamura Y. Noguchi M. Leland P. Friedmann M.C. Miyajima A. Puri R.K. Paul W.E. Leonard W.J. Science. 1993; 262: 1880-1883Crossref PubMed Scopus (740) Google Scholar), we explored further the specificity of hIL13-PE38QQR action on A431 and CRL1739 cells, the two cell lines with different sensitivities to the chimeric toxin (Fig. 4). The cells were treated with hIL13-PE38QQR with or without rhIL2 (a gift from Cetus) at a concentration of 1.0 or 10 μg/ml. The rhIL2 did not have any blocking action on hIL13-PE38QQR on the two cell lines, even at 10,000-fold molar excess over the chimeric toxin (Fig. 4). These results indicate that the cell killing by the hIL13-toxin is independent of the presence of hIL2.Figure 4:Failure to block the cytotoxicity of hIL13-PE38QQR on cancer cells by 1.0 μg/ml or 10 μg/ml of hIL2. The dashed line shows 50% of [3H]leucine incorporation. ● hIL13-PE38QQR; □, with hIL2 (1 μg/ml); Δ, with hIL2 (10 μg/ml).View Large Image Figure ViewerDownload Hi-res image Download (PPT)hIL4, Unlike hIL2, Blocks the Action of hIL13-PE38QQRWe also added native hIL4 to cells and then treated with hIL13-PE38QQR, as another measure of specificity (Fig. 5). Unexpectedly, we found that hIL4 inhibited very potently the cytotoxic activity of the hIL13-toxin. This phenomenon was seen on all of the tested cell lines, which includes Colo201, A431 (Fig. 5), and CRL1739.2 2W. Debinski, unpublished data. To investigate the possibility that hIL13 and hIL4 may compete for the same binding site, we also treated the cells with hIL4-based recombinant toxin, hIL4-PE38QQR (15Debinski W. Puri R.K. Pastan I. Int. J. Cancer. 1994; 58: 744-748Crossref PubMed Scopus (22) Google Scholar) (Fig. 5). The cytotoxic action of hIL4-PE38QQR had already been shown to be blocked by an excess of hIL4 but not of hIL2(15Debinski W. Puri R.K. Pastan I. Int. J. Cancer. 1994; 58: 744-748Crossref PubMed Scopus (22) Google Scholar). In the present experiment, we examined hIL13 and found that it blocked potently the cytotoxic activity of hIL4-PE38QQR (Fig. 5). Also, the action of another hIL4-based chimeric toxin, hIL4-PE4E(12Debinski W. Puri R.K. Kreitman R.J. Pastan I. J. Biol. Chem. 1993; 268: 14065-14070Abstract Full Text PDF PubMed Google Scholar), was blocked by an excess of hIL13 on Colo201 and A431 cells.2 Thus, the cytotoxicity of hIL13-PE38QQR is blocked by an excess of hIL13 or hIL4, and the cytotoxic action of hIL4-PE38QQR is also blocked by the same two growth factors. However, IL2 does not block the action of either chimeric toxin. These results strongly suggest that hIL4 and hIL13 have affinities for the same binding site.Figure 5:Blocking the cytotoxicity of hIL13-PE38QQR by hIL4 and blocking the cytotoxicity of hIL4-PE38QQR by hIL13 on cancer cell lines. hIL4 was added at a concentration of 0.25 μg/ml and hIL13 at a concentration of 1.0 μg/ml. The dashed line shows 50% of [3H]leucine incorporation. ● hIL13-PE38QQR; ○, with hIL4; ■, hIL4-PE38QQR; □, with hIL13.View Large Image Figure ViewerDownload Hi-res image Download (PPT)This conclusion was supported by the observation of one cytokine blocking the effect of a mixture of the two chimeric toxins. Fig. 6 illustrates experiments in which we used both hIL13- and hIL4-PE38QQR at equimolar concentrations on A431 cells. When the cells were incubated with both chimeric toxins concomitantly, the cytotoxic action was preserved, and additive effect was observed as expected.2 An excess of hIL13 efficiently blocked the action of a mixture of the two chimeric toxins (Fig. 6). Moreover, neither hIL13 (Fig. 6) nor hIL42 blocked the cell killing by another mixture composed of hIL13-PE38QQR and TGFα-PE40, a chimeric toxin that targets the epidermal growth factor receptor (TGFα-based chimeric toxin, TGFα-PE40)(21Siegall C.B. Xu Y.-H. Chaudhary V.K. Adhya S. FitzGerald D.J.P. Pastan I. FASEB J. 1992; 3: 2647-2652Crossref Scopus (70) Google Scholar). The same was observed on Colo201 cells.2Figure 6:hIL13 blocks the cytotoxic action of a mixture of hIL13-PE38QQR and hIL4-PE38QQR, but it does not block the cytotoxicity of a mixture of hIL13-PE38QQR and TGFα-PE40. The dashed line shows 50% of [3H]-leucine incorporation. Δ, with hIL13.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Blocking the Action of Chimeric Toxins by the Cytokines Is Due to the Competition for Binding SitesIt was important to determine whether the binding of hIL4 chimeric toxin is affected by hIL13, since receptor binding is the first step in the cytotoxic action of chimeric protein. To investigate this, we performed competitive binding assays. The iodination of hIL4-PE38QQR and the binding experiments were carried out as described under “Experimental Procedures.” hIL4-PE38QQR competed for the binding of 125I-hIL4-PE38QQR to A431 cells with an apparent IC50 of 4 × 10-8M (Fig. 7). In addition, hIL13 also competed for the 125I-hIL4-PE38QQR binding site with a comparable potency to that exhibited by the chimeric protein. More extensive binding studies have shown that hIL13 competes for hIL4 binding sites on human renal carcinoma cell lines also(8Obiri N.I. Debinski W. Leonard W.J. Puri R.K. J. Biol. Chem. 1995; 270: 8797-8804Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar).Figure 7:Competitive binding assay on A431 cells. Data are expressed as a percentage of total 125I-hIL4-PE38QQR binding to cells. The points are the average of experiments performed in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We excluded the possibility of an influence of hIL13 or hIL4 on the process of receptor-mediated endocytosis and post-binding PE cellular toxicity steps by adding the following to cells: (i) native PE (PE binds to the α2-macroglobulin receptor), (ii) TGFα-PE40, and (iii) a recombinant immunotoxin C242rF(ab′)-PE38QQR(20Debinski W. Pastan I. Clin. Cancer Res. 1995; (in press)Google Scholar). C242rF(ab′)-PE38QQR binds a tumor-associated antigen that is a sialylated glycoprotein(19Debinski W. Karlsson B. Lindholm L. Siegall C.B. Willingham M.C. FitzGerald D. Pastan I. J. Clin. Invest. 1992; 90: 405-411Crossref PubMed Scopus (39) Google Scholar). We observed the expected potent cytotoxic actions of these recombinant toxins and neither hIL13 nor hIL4 blocked these actions on A431 and Colo205 cells.2hIL4 and hIL13 Compete for a Common Binding Site on Carcinoma Cells but May Evoke Different Biological EffectsDespite being competitors for the same binding site, some differences were observed in hIL13- and hIL4-induced cellular effects. Protein synthesis was inhibited in A431 epidermoid carcinoma cells in a dose-dependent manner by hIL4 alone (Fig. 8) or by a ADP ribosylation-deficient chimeric toxin containing hIL4(15Debinski W. Puri R.K. Pastan I. Int. J. Cancer. 1994; 58: 744-748Crossref PubMed Scopus (22) Google Scholar). This effect of hIL4 or enzymatically deficient chimeric toxin can be best seen with a prolonged time of incubation (≥24 h) and requires concentrations of hIL4 many fold higher than that of the active chimeric toxin in order to cause a substantial decrease in tritium incorporation. When, however, A431 cells were treated with various concentrations of hIL13, no inhibition (or stimulation) of protein synthesis was observed, even at concentrations as high as 10 μg/ml of hIL13 for a 72-h incubation (Fig. 8). The same lack of response to hIL13 was found on renal cell carcinoma cells PM-RCC.3 3R. K. Puri, unpublished data. Thus, hIL13 and hIL4 possess a common site but may transduce differently in carcinoma cells expressing this common site, such as A431 and PM-RCC cells.Figure 8:hIL4, but not hIL13, inhibits protein synthesis in A431 epidermoid carcinoma cells. Data represent the average of quadruplicates. Standard deviations are marked as vertical bars.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONWe have made a novel recombinant chimeric toxin, hIL13-PE38QQR, composed of hIL13 and a mutated PE, PE38QQR. This chimeric toxin exerts a potent cytotoxic activity on a spectrum of human carcinomas. The cytotoxic activity of hIL13-PE38QQR, which can only occur subsequent to the internalization of a ligand-receptor complex(16Pastan I. Chaudhary V.K. FitzGerald D. Annu. Rev. Biochem. 1992; 61: 331-354Crossref PubMed Scopus (311) Google Scholar), is competed for by hIL13 and also by hIL4. Thus, there is a common internalized binding site for the two cytokines on many human carcinoma cells.Our present results add new information on the interrelatedness between receptors for IL13 and IL4, since we demonstrate unequivocally that the wild-type hIL