A model for human lymphocyte ontogeny has been developed in a normal mouse. Human bone marrow, depleted of mature T and B lymphocytes, and bone marrow from mice with severe combined immunodeficiency were transplanted into lethally irradiated BALB/c mice. Human B and T cells were first detected 2 to 4 months after transplantation and persisted for at least 6 months. Most human thymocytes (30 to 50 percent of total thymocytes) were CD3 + CD4 + CD8 + . Human immunoglobulin was detected in some chimeras, and a human antibody response to dinitrophenol could be generated after primary and secondary immunization.
Four ErbB receptors and multiple growth factors sharing an epidermal growth factor (EGF) motif underlie transmembrane signaling by the ErbB family in development and cancer. Unlike other ErbB proteins, ErbB-2 binds no known EGF-like ligand. To address the existence of a direct ligand for ErbB-2, we applied algorithms based on genomic and cDNA structures to search sequence data bases. These searches reidentified all known EGF-like growth factors including Epigen (EPG), the least characterized ligand, but failed to identify novel factors. The precursor of EPG is a widely expressed transmembrane glycoprotein that undergoes cleavage at two sites to release a soluble EGF-like domain. A recombinant EPG cannot stimulate cells singly expressing ErbB-2, but it acts as a mitogen for cells expressing ErbB-1 and co-expressing ErbB-2 in combination with the other ErbBs. Interestingly, soluble EPG is more mitogenic than EGF, although its binding affinity is 100-fold lower. Our results attribute the anomalous mitogenic power of EPG to evasion of receptor-mediated depletion of ligand molecules, as well as to inefficient receptor ubiquitylation and down-regulation. In conclusion, EPG might represent the last EGF-like growth factor and define a category of low affinity ligands, whose bioactivity differs from the more extensively studied high affinity ligands. Four ErbB receptors and multiple growth factors sharing an epidermal growth factor (EGF) motif underlie transmembrane signaling by the ErbB family in development and cancer. Unlike other ErbB proteins, ErbB-2 binds no known EGF-like ligand. To address the existence of a direct ligand for ErbB-2, we applied algorithms based on genomic and cDNA structures to search sequence data bases. These searches reidentified all known EGF-like growth factors including Epigen (EPG), the least characterized ligand, but failed to identify novel factors. The precursor of EPG is a widely expressed transmembrane glycoprotein that undergoes cleavage at two sites to release a soluble EGF-like domain. A recombinant EPG cannot stimulate cells singly expressing ErbB-2, but it acts as a mitogen for cells expressing ErbB-1 and co-expressing ErbB-2 in combination with the other ErbBs. Interestingly, soluble EPG is more mitogenic than EGF, although its binding affinity is 100-fold lower. Our results attribute the anomalous mitogenic power of EPG to evasion of receptor-mediated depletion of ligand molecules, as well as to inefficient receptor ubiquitylation and down-regulation. In conclusion, EPG might represent the last EGF-like growth factor and define a category of low affinity ligands, whose bioactivity differs from the more extensively studied high affinity ligands. Growth factors play important roles in developmental cell lineage determination and in tissue remodeling throughout adulthood. One of the most extensively studied families of growth factors comprises polypeptides sharing an epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; CHO, Chinese hamster ovary; EPG, Epigen; EPR, Epiregulin; IL-3, interleukin-3; NRG, Neuregulin; TGFα, transforming growth factor α; HB, heparin-binding; HA, hemagglutinin; EndoH, endoglycosidase H; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PNGaseF, peptide N-glycosidase F; E3, ubiquitin-protein isopeptide ligase. motif (reviewed in Ref. 1Stein R.A. Staros J.V. J. Mol. Evol. 2000; 50: 397-412Crossref PubMed Scopus (79) Google Scholar). This highly conserved motif, which includes six canonical cysteines, binds to a family of four cell surface receptors, called ErbB (or HER) proteins. The extracellular domain of each ErbB protein displays binding specificity to several EGF-like ligands, which activate the intracellular tyrosine kinase. Notably, ErbB-3 binds several types of neuregulins, but its tyrosine kinase domain is catalytically inactive (2Guy P.M. Platko J.V. Cantley L.C. Cerione R.A. Carraway III, K.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8132-8136Crossref PubMed Scopus (596) Google Scholar). Conversely, ErbB-2 binds with no known ligand directly but is transactivated through heterodimerization with other ErbBs (3Klapper L.N. Glathe S. Vaisman N. Hynes N.E. Andrews G.C. Sela M. Yarden Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4995-5000Crossref PubMed Scopus (369) Google Scholar). Interestingly, ErbB-2 is widely expressed in a dynamic and heterogeneous manner. An example is provided by human tumors of mammary and ovarian origins; because of gene amplification, ErbB-2 is overexpressed in a subset of relatively aggressive tumors (4Slamon D.J. Godolphin W. Jones L.A. Holt J.A. Wong S.G. Keith D.E. Levin W.J. Stuart S.G. Udove J. Ullrich A. Press M.F. Science. 1989; 244: 707-712Crossref PubMed Scopus (6261) Google Scholar). The apparent absence of a bone fide ligand for ErbB-2 is also relevant to another route of ErbB-mediated oncogenesis, namely increased production of a growth factor where autocrine ligand secretion drives proliferation of various cancers (reviewed in Ref. 5Salomon D.S. Brandt R. Ciardiello F. Normanno N. Crit. Rev. Oncol. Hematol. 1995; 19: 183-232Crossref PubMed Scopus (2451) Google Scholar). Because ErbB-2 is a widely expressed oncoprotein and antibodies directed at this surface antigen can prolong survival of breast cancer patients (6Baselga J. Tripathy D. Mendelsohn J. Baughman S. Benz C.C. Dantis L. Sklarin N.T. Seidman A.D. Hudis C.A. Moore J. Rosen P.P. Twaddell T. Henderson I.C. Norton L. J. Clin. Oncol. 1996; 14: 737-744Crossref PubMed Scopus (1268) Google Scholar), it is crucial to examine the contention that ErbB-2 functions as a ligand-less co-receptor, which enhances and prolongs signaling by autocrine or stroma-derived EGF-like ligands (reviewed in Ref. 7Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell. Biol. 2001; 2: 127-137Crossref PubMed Scopus (5633) Google Scholar). The ErbB family presents another open question that relates to the multiplicity of EGF-like ligands sharing specificity to the same receptor (reviewed by Ref. 8Riese II, D.J. Stern D.F. Bioessays. 1998; 20: 41-48Crossref PubMed Scopus (698) Google Scholar). Thus, along with EGF and the transforming growth factor α (TGFα), ErbB-1 binds the heparin-binding EGF-like growth factor (HB-EGF), Betacellulin, Amphiregulin (AR), Epiregulin, and Epigen (9Strachan L. Murison J.G. Prestidge R.L. Sleeman M.A. Watson J.D. Kumble K.D. J. Biol. Chem. 2001; 276: 18265-18271Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Likewise, several ligands that bind ErbB-4 also bind ErbB-1, with the exception of NRG-3 and NRG-4, which bind ErbB-4 exclusively. Genetic manipulation of EGF-like ligands in mammals assigned partially overlapping but distinct functions to individual growth factors, in processes as diverse as mammary, cardiac and intestinal development, and wound healing (see for example Ref. 10Troyer K.L. Luetteke N.C. Saxon M.L. Qiu T.H. Xian C.J. Lee D.C. Gastroenterology. 2001; 121: 68-78Abstract Full Text PDF PubMed Scopus (93) Google Scholar). These and other lines of evidence imply that ligand multiplicity reflects functional features unique to each EGF-like growth factor. The present study addressed the multiplicity of EGF-like molecules and the possibility that mammalian genomes encode a yet uncharacterized ligand for ErbB-2. By designing a genome-wide screen that can identify ErbB ligands on the basis of their intron-exon organization, we reidentified all known ligands of ErbB proteins but found no novel molecule that can potentially qualify as an ErbB-2 ligand. Concentrating on the least characterized member of the EGF family, namely Epigen (EPG), we highlight several biochemical and physiological attributes that distinguish low affinity ligands from the extensively characterized high affinity family members. Bioinformatics and Phylogenetics—The approximate exon-intron boundary for each EGF-like gene was determined (using tblastn) by comparison of the protein sequence for each gene with the corresponding genomic locus. For the purpose of the data base searches performed herein, the EGF domain was defined as that encoding the six invariant cysteine residues of the domain and the additional five flanking amino acids both N- and C-terminally to cysteine 1 and cysteine 6, respectively. Two separate multiple alignments were generated for sequences encoding Exon-1 and Exon-2, which were then compiled into two independent profiles (Profilemake; GCG10). The profiles were used as criteria for a stepwise Smith-Waterman-based search of human genomic data (Biocellerator device, Compugen, Israel; HTGS and NR data bases). The first 2000 hits from the Exon-1 profile search were saved and recompiled into a new, miniature data base using PERL script. These 2000 sequences were then scanned, this time using the Exon-2 profile, to identify EGF-like ligands. The human Epigen-Epiregulin genomic locus was extracted from a data base provided by Celera Genomics. Their localization to chromosome 4q21.21 was assigned with the aid of NCBI MapView (version 34). Phylogenetic data were generated with the aid of ClustalX (version 1.81; default settings) and visualized with a modification of an output generated from TREEVIEW (11Page R.D.M. Comput. Appl. Biosci. 1996; 12: 357-358PubMed Google Scholar). Expression of Pro-Epigen in Mammalian Cells—The coding sequence of pro-EPG was amplified using PCR from an IMAGE clone (Gen-Bank™; BU540655) and cloned into pIRES-puro vector (12Hobbs S. Jitrapakdee S. Wallace J.C. Biochem. Biophys. Res. Commun. 1998; 252: 368-372Crossref PubMed Scopus (222) Google Scholar), with or without a C-terminal HA tag. The plasmid was transfected into CHO cells, and stable clones were selected with 10 μg/ml puromycin. Immunoprecipitates of anti-HA (Roche Applied Science) and anti-EPG antibodies were equilibrated with 50 mm citrate (pH 5.5) or saline for deglycosylation using endoglycosidase H (EndoH) or peptide N-glycosidase F, respectively. Deglycosylation was performed at 37 °C for 2 h. The proteins were separated on a 16.5% Tricine gel and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). To biotinylate cell surface proteins, the cell monolayers were washed with saline and incubated with biotin-N-hydroxysuccinimidyl ester (Pierce) for 1 h on ice. Excess biotin was quenched using 100 mm glycine in saline, and the cells were washed extensively and incubated at 37 °C. Expression and Purification of Recombinant Growth Factors—The EGF-like domain, including five residues N-terminal to cysteine 1 and five and eight residues C-terminal to cysteine 6 of human NRG-β1 and human EPG, respectively, was cloned into the pET32b vector and expressed as C-terminal thioredoxin fusion proteins with the Factor Xa cleavage site adjacent to the N-terminal residue of the EGF-like domain. The proteins were expressed in Escherichia coli (BL21) using standard procedures. Expressed proteins were first purified on a nickel-nitrilotriacetic acid column, cleaved using Factor-Xa (New England Biolabs, Beverly, MA), and then purified on a Superdex-30 (Amersham Biosciences) column. Generation and Affinity Purification of an Anti-EPG Antibody—New Zealand rabbits were immunized with a purified EGF-like domain of EPG (0.1 mg). For affinity purification, a recombinant EPG fusion protein was immobilized on cyanogen bromide-activated Sepharose 4B (Amersham Biosciences) according to the manufacturer's protocol. The serum was diluted 10-fold using column buffer (Tris-HCl, pH 8.0, 250 mm NaCl) and passed through the column. The column was then washed extensively, and bound proteins were eluted using 100 mm glycine-HCl (pH 2.5). Cell Proliferation and Stimulation Assays—A previously described method (13Mosman T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (46504) Google Scholar) has been used with the following modifications: 5 × 104 32D cells were plated in triplicate in a 96-well plate and treated with different concentrations of ligand or with a fixed ligand concentration (100 ng/ml) for different time intervals. In experiments with extended periods of treatment, the cells were plated at a density of 1000 cells/well of a 96-well plate. The cells were harvested at each time point and incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.1 mg/ml) for 2 h. Thereafter, the cells were extracted with acidified 2-propanol, and the optical density was measured at 570 nm with a reference 650 nm filter. For receptor stimulation, 32D cells were washed extensively in medium devoid of interleukin-3 (IL-3) and divided equally. The cells were then stimulated with various ligands for different periods of time, collected by centrifugation, and lysed in lysis buffer, and equal amounts of lysate were separated using gel electrophoresis. Separated proteins were then transferred onto nitrocellulose membranes and probed with specific antibodies. Ligand Binding Assays—Ligands were radiolabeled using Iodogen (Pierce) following the manufacturer's protocol. Cells grown in 48-well plates were washed with ice-cold binding buffer (Dulbecco's modified Eagle's medium containing 20 mm HEPES, pH 7.5, and 0.1% albumin) and incubated on ice for 3 h with different ligand concentrations in duplicates. Thereafter, media were aspirated, cells were washed and extracted in 0.1% sodium dodecylsulfate and 0.1 n NaOH, and cell-associated radioactivity was determined. Ligand Degradation Assay—Cos-7 cells were equally plated in a 48-well plate, and the experiment was performed in duplicates. Non-specific binding was determined with 100-fold excess unlabeled EGF. The cells were washed, equilibrated with binding buffer at 37 °C, and incubated with radiolabeled ligands at 37 °C. After incubation, the medium was collected, and intact ligand in the medium was precipitated using trichloroacetic acid. The cells were washed once with warm binding buffer, and this was added to the medium fraction. The cells were then transferred to ice and incubated with cold acid wash buffer (50 mm glycine-HCl, pH 3.0, 100 mm NaCl, 2 mg/ml polyvinylpyrrolidone). The wash buffer was collected, and radioactivity associated with this sample was considered as the surface-bound ligand. Lysates of the cells were also prepared. The sum of radioactivity obtained in the acid-precipitable and acid wash fractions was considered as intact extracellular ligand. Sensitivity of Ligand Binding to pH—A431 cells were plated equally in 48-well plates, and the experiment was performed in duplicates. After 24 h, the cells were moved to ice and equilibrated with 50 mm phosphate buffers at different pH levels (7.5-6.0) containing 100 mm NaCl and 0.1% albumin. The equilibrated cells were then incubated for 2 h on ice with ligand-containing solutions buffered at various pH levels. At the end of the incubation, the cells were washed twice with buffers at the respective pH level. The cells were lysed, and associated radioactivity was measured using a γ-counter. Receptor Ubiquitylation Assay—Cos-7 cells were transiently transfected with the indicated plasmids using Lipofectamine (Invitrogen). Forty-eight hours later, the cells were stimulated with EGF or EPG for 10 min. ErbB-1 was immunoprecipitated from cell lysates, or the lysate was directly resolved by electrophoresis. Separated proteins were then transferred onto a nitrocellulose membrane and probed with antibodies to HA (Roche Applied Science), ErbB-1 (Santa Cruz Biotechnology, Santa Cruz, CA), or c-Cbl (Santa Cruz). Receptor Down-regulation Assay—Cos-7 cells were transfected with plasmids encoding ErbB-1, along with a c-Cbl plasmid as indicated. Twenty-four hours later, the cells were divided into 6-well plates, and after another 24 h the cells were stimulated with various ligands at 37 °C. At the end of each time interval, the plates were placed on ice, surface-bound ligand was stripped using acid wash buffer (150 mm acetic acid and 150 mm NaCl), and the cells were incubated for 2 h with 125I-EGF (5 ng/ml) in binding buffer. Following incubation, the cells were washed using binding buffer, and cell-associated radioactivity was determined. Immunohistochemistry Using Anti-EPG Antibodies—Murine tissues were isolated, frozen in powdered dry ice, and embedded in paraffin. Staining was performed on 10-μm thick paraffin sections after an antigen retrieval procedure using 10 mm citric acid (pH 6.0). Tissue sections were first blocked using normal serum and then incubated with the anti-EPG antibody (0.5 μg/ml) or anti-TGFα (BioTope). Visualization was achieved using the Vectastain ABC system (Vectorlabs, Burlingham, CA) or donkey anti-rabbit and anti-rat fluorescent antibodies (Jackson ImmunoResearch, West Grove, PA). Human tissue sections were stained with the anti-EPG antibody using a similar protocol. PCR Analysis of EPG Expression—Prostate cancer xenografts were grown in castrated (androgen-independent) and noncastrated (androgen-dependent) nude mice, as described (14Pinthus J.H. Waks T. Schindler D.G. Harmelin A. Said J.W. Belldegrun A. Ramon J. Eshhar Z. Cancer Res. 2000; 60: 6563-6567PubMed Google Scholar). Total RNA was isolated from 0.1 mg of each tissue using TRIzol (Sigma). Primers for PCR were selected within the coding region of EPG using the web interface of the Primer 3 software (MIT.EDU). The reverse transcriptase reaction with a sample of total RNA was performed using Superscript (Invitrogen), and PCRs were performed using BioTaq (Bioline, London, UK) for 30 cycles. A second round of PCR was performed to increase the amount of product, and the samples were divided into two. One part was left untreated, and the other was digested with NdeI. Cell Differentiation and Aortic Ring Assays—Differentiation of mammary cells has been described before (15Bacus S.S. Gudkov A.V. Zelnick C.R. Chin D. Stern R. Stancovski I. Peles E. Ben Baruch N. Farbstein H. Lupu R. Cancer Res. 1993; 53: 5251-5261PubMed Google Scholar). PC12 cells stably expressing ErbB-4 (16Vaskovsky A. Lupowitz Z. Erlich S. Pinkas-Kramarski R. J. Neurochem. 2000; 74: 979-987Crossref PubMed Scopus (82) Google Scholar) were plated in 24-well, collagen-coated plates and treated with the indicated ligands (100 ng/ml) for 7 days. Normal human prostate epithelial cells, RWPE-1, were plated on growth factor-depleted Matrigel at a density of 1000 cells/well in a 96-well plate. The cells were incubated with KSFM medium (Invitrogen) (17Bello D. Webber M.M. Kleinman H.K. Wartinger D.D. Rhim J.S. Carcinogenesis. 1997; 18: 1215-1223Crossref PubMed Scopus (330) Google Scholar) containing growth factors at 100 ng/ml for 5 days, and the medium was replenished after 3 days. Aortas from 6-10-week-old mice were isolated, cleaned of surrounding tissue, and washed with saline. Each aorta was cut into 1-mm-thick rings and embedded in collagen (isolated from rat tail). The rings were then incubated in medium, either with or without growth factors (at 100 ng/ml). The medium was changed every 3 days, and the rings were monitored for up to 14 days. To visualize microvessels, the rings were fixed overnight using 4% formaldehyde in saline, stained for 4 h with a solution containing 0.02% crystal violet in 50% ethanol, and subsequently destained. A Genome-wide Screen Reidentified All Known EGF-like Molecules, Including the Most Recent Addition, Epigen—Previously we have reported the discovery of NRG-4 using a bioinformatics approach (18Harari D. Tzahar E. Romano J. Shelly M. Pierce J.H. Andrews G.C. Yarden Y. Oncogene. 1999; 18: 2681-2689Crossref PubMed Scopus (255) Google Scholar), where the search criteria were based on the six conserved cysteine residues of the EGF-like domain. Repeated searches of the expanded expressed sequence tag data base detected no novel ligands, which led us to examine alternative strategies. Analyses of the genomic structure of the known ErbB ligands revealed that the EGF-like domain of all the mammalian ligands are encoded by two exons (defined here as Exon-1 and Exon-2), where Exon-1 encodes four of the N-terminal cysteine residues, and Exon-2 encodes cysteines 5 and 6 (Fig. 1A). NRG1 and NRG2 genes harbor a supplementary Exon-2, and through alternative splicing, Exon-1 is fused to either the first or the second Exon-2, thereby generating the α or β isoforms. Interestingly, the EGF-like domain of Lin-3, the single EGF-like growth factor in Caenorhabditis elegans, is encoded by three exons, but the five corresponding genes of Drosophila display three different genomic patterns in the EGF-like domains, including one (vein) that resembles the mammalian pattern (Fig. 1A). The conserved genomic organization of the different mammalian ErbB ligands provided a means in which a stepwise Smith-Waterman-based search could be employed to screen for new EGF-like ligands from genomic data. Unlike gapped BLAST searches, Smith-Waterman-based searches are unable to accommodate large gaps (e.g. intron sequences) when performing sequence alignments. Hence, the search procedure was performed as follows. First, two separate multiple sequence alignments were generated corresponding to the segments of the EGF-like domains encoded by Exon-1 and Exon-2 for all known ligands. These alignments were then converted into two sequence profiles. The profile of protein sequences encoded by Exon-1 was then used as a bait to perform a Smith-Waterman-based profile search against genomic sequence data (detailed under "Materials and Methods"). The first 2000 hits from this search were recompiled into a miniature data base. This data base was then searched once again using the sequence profile generated from Exon-2. All hits harboring both Exon-1- and Exon-2-encoding sequences were thus identified. Using this methodology, we detected all of the ErbB ligands for which genomic sequences are available (all ligands but Betacellulin), including the most recent addition, namely Epigen (9Strachan L. Murison J.G. Prestidge R.L. Sleeman M.A. Watson J.D. Kumble K.D. J. Biol. Chem. 2001; 276: 18265-18271Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). To test the power of the genomic search, we created a series of sequence profiles, each lacking a single ErbB ligand and used them to rescreen the genomic and expressed sequence tag data bases. In each case, the missing ligand was reidentified from the genomic data, demonstrating that the search strategy is robust. Hence, we concluded that the combined expressed sequence tag and genomic search strategies are exhaustive, with no novel ligands being identified. Alignment of the EGF-like motifs of all human ErbB ligands indicated that EPG is most similar to EPG. A phylogeny analysis clustered five ligands of ErbB-1 and revealed an Epigen-Epiregulin relationship (Fig. 1B). Because of the small length of the EGF domain and the high disparity of sequence identity between different members of the family, the significance of the Epigen-Epiregulin clustering is not particularly high (a boot-strap value of 398 of 1000 trials). Nevertheless, in support of relatedness, EPG and EPR co-localize to the same genomic locus, at chromosomal position 4q21, and their open reading frames are separated by a mere 25 kilobase pairs (Fig. 1C). Similarly, this genomic linkage is evident in mice (data not shown), demonstrating that the Epiregulin-Epigen microchromosomal duplication took place in mammals before the evolutionary diversion of primates and rodents. Pro-Epigen Is a Transmembrane Glycoprotein Processed at Two Proteolytic Sites—Analysis of the primary sequence of pro-EPG predicts a type-1 transmembrane protein (9Strachan L. Murison J.G. Prestidge R.L. Sleeman M.A. Watson J.D. Kumble K.D. J. Biol. Chem. 2001; 276: 18265-18271Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), but no data are available on glycosylation, proteolytic processing, or association with the plasma membrane. To address these issues, we stably expressed in CHO cells full-length pro-EPG, with and without a HA peptide tag at the C terminus. In addition, we immunized rabbits with recombinant EPG expressed in bacteria (rEPG, see below) and affinity-purified the antiserum. The immune serum and the purified antibody specifically recognized three proteins (p40, p30, and p13) in pro-EPG-expressing cells (Fig. 2A). Labeling of the surface of pro-EPG-expressing CHO cells with biotin detected p40 and p13 (Fig. 2B), indicating that p30 is an intracellular protein and confirming delivery of pro-EPG to the plasma membrane. To further prove that p40 is expressed at the cell surface, we tested the acquisition of resistance to EndoH, which parallels translocation of EndoH-sensitive high mannose precursors from the endoplasmic reticulum to the Golgi apparatus. Separately, immunoprecipitates of EPG were treated with peptide N-glycosidase F (PNGaseF), an enzyme that cleaves all N-linked glycans but core fucosylated chains. Biotin labeled pro-EPG (p40) was susceptible to PNGaseF but resistant to EndoH (Fig. 2B), indicating that modifications of asparagine-linked oligosaccharides of pro-EPG is completed in the Golgi, prior to delivery to the cell surface. Unlike p40, p13 displayed resistance to both EndoH and PNGaseF, and p30 was resistant to EndoH but sensitive to PNGaseF (Fig. 2, B and C). In addition to p13, p30, and p40, anti-HA antibodies detected a fourth protein, p7, which displayed resistance to both enzymes. On the basis of these results, and the presence of two predicted asparagine-based glycosylation sites, we inferred that pro-EPG carries an N-terminal N-glycosylation domain and two putative cleavage sites (Fig. 2D). To test this model, we surface-labeled pro-EPG-expressing cells with biotin, quenched reactive biotin, and chased cells in fresh medium. The most prominently labeled species was p13, suggesting that cleavage at the N-terminal site of pro-EPG precedes proteolysis at the membrane-flanking site (probably an Ala103-Val motif, similar to other EGF-like ligands; Fig. 2E). In summary, following insertion in the plasma membrane, the precursor of EPG undergoes two proteolytic cleavage events; a relatively rapid event releases the glycosylated domain, and a subsequent event clips within the short segment connecting the EGF-like domain with the transmembrane helix. Epigen Cannot Stimulate ErbB-2 in Isolation—EPG is the least characterized EGF-like ligand (9Strachan L. Murison J.G. Prestidge R.L. Sleeman M.A. Watson J.D. Kumble K.D. J. Biol. Chem. 2001; 276: 18265-18271Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), and its interaction with ErbB-2 has not been investigated. Therefore, we expressed EPG, as well as NRG-1β for control, as thioredoxin fusion proteins in bacteria, cleaved the products to remove thioredoxin, and purified the recombinant growth factors to homogeneity. Radiolabeling of rEPG confirmed absence of trace impurities (Fig. 3A), and mass spectrometry yielded a molecular mass consistent with oxidation of all cysteines. Next, we tested whether EPG could activate ErbB-2 in the absence of other members of the ErbB family. The D2 derivative of the IL-3-dependent 32D myeloid cell line, stably expressing human ErbB-2 on an ErbB-null background, was used (19Pinkas-Kramarski R. Soussan L. Waterman H. Levkowitz G. Alroy I. Klapper L. Lavi S. Seger R. Ratzkin B.J. Sela M. Yarden Y. EMBO J. 1996; 15: 2452-2467Crossref PubMed Scopus (698) Google Scholar). Although IL-3 supported robust cell proliferation, treatment of D2 cells with rEPG in the absence of IL-3 resulted in growth arrest and partial cell death (Fig. 3B). These results indicate that EPG, like its closest homologue, EPR (20Shelly M. Pinkas-Kramarski R. Guarino B.C. Waterman H. Wang L.M. Lyass L. Alimandi M. Kuo A. Bacus S.S. Pierce J.H. Andrews G.C. Yarden Y. J. Biol. Chem. 1998; 273: 10496-10505Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), and in fact all other members of the EGF family, is unable to stimulate ErbB-2 in the absence of other ErbBs. Epigen Is a Potent Activator of ErbB-1 Despite Low Binding Affinity—Next we analyzed the activity of EPG toward the other ErbB receptors. We used derivatives of the 32D cell line expressing ErbB-1 (D1), ErbB-3 (D3), or ErbB-4 (D4), either singly or in combination with ErbB-2 (D12, D23, and D24). When D1 cells were treated with EGF, TGFα, and EPG over 4 days, EPG was found to be the most potent mitogen in comparison with the other ligands (Fig. 3C). Under similar experimental conditions neither D3 nor D4 cells displayed significant responses to EPG (data not shown). In line with the notion that ErbB-2 enhances signaling by the other ErbB receptors, the induction of proliferation observed in EPG-treated D12 cells was higher than in D1 cells (Fig. 3, C and D), and both D23 and D24 cells showed increases in cell proliferation, as reflected in both kinetics and dose curves (Fig. 3, E and F). Stimulation of the 32D cell lines with EGF or NRG-1β induced robust receptor phosphorylation, whereas the activity of EPG was relatively weak (Fig. 4, A and B, and data not shown). However, in the case of D1 and D12 cells, where EPG induces relatively potent mitogenic responses, the weak EPG-induced receptor phosphorylation was sustained up to 45 min, and extracellular signal-regulated kinase/mitogen-activated protein kinase signals were almost comparable with those induced by EGF (Fig. 4, A and B). To understand what bearing the binding affinity of EPG has on its relatively high mitogenicity, ligand displacement analyses were performed. Unexpectedly, displacement of a radiolabeled EGF by EPG indicated an ∼100-fold difference in binding affinity, in favor of EGF (Fig. 4C, left panel). The reciprocal assay, which used a radiolabeled EPG and an unlabeled EGF, corroborated this finding (Fig. 4C, right panel). Further, the bi-sigmoidal curve observed
Tumor-treating fields (TTFields) are a localized, antitumoral therapy using alternating electric fields, which impair cell proliferation. Combining TTFields with tumor immunotherapy constitutes a rational approach; however, it is currently unknown whether TTFields' locoregional effects are compatible with T cell functionality. Healthy donor PBMCs and viably dissociated human glioblastoma samples were cultured under either standard or TTFields conditions. Select pivotal T cell functions were measured by multiparametric flow cytometry. Cytotoxicity was evaluated using a chimeric Ag receptor (CAR)-T-based assay. Glioblastoma patient samples were acquired before and after standard chemoradiation or standard chemoradiation + TTFields treatment and examined by immunohistochemistry and by RNA sequencing. TTFields reduced the viability of proliferating T cells, but had little or no effect on the viability of nonproliferating T cells. The functionality of T cells cultured under TTFields was retained: they exhibited similar IFN-γ secretion, cytotoxic degranulation, and PD1 upregulation as controls with similar polyfunctional patterns. Glioblastoma Ag-specific T cells exhibited unaltered viability and functionality under TTFields. CAR-T cells cultured under TTFields exhibited similar cytotoxicity as controls toward their CAR target. Transcriptomic analysis of patients' glioblastoma samples revealed a significant shift in the TTFields-treated versus the standard-treated samples, from a protumoral to an antitumoral immune signature. Immunohistochemistry of samples before and after TTFields treatment showed no reduction in T cell infiltration. T cells were found to retain key antitumoral functions under TTFields settings. Our data provide a mechanistic insight and a rationale for ongoing and future clinical trials that combine TTFields with immunotherapy.
The goal of this study was to examine the use of diffusion-weighted magnetic resonance imaging (DW-MRI) for the assessment of early progression of photodamage induced by Pd-bacteriopheophorbide (TOOKAD)-based photodynamic therapy (PDT). TOOKAD is a novel second-generation photosensitizer for PDT of solid tumors developed in our laboratory and presently under clinical trials for prostate cancer (PC) therapy. Using the subcutaneous human prostate adenocarcinoma WISH-PC14 xenografts in nude mice as a model, a unique biphasic change in the apparent diffusion coefficient (ADC) was observed within the first 24 hours post-PDT, with initial decrease followed by an increase in ADC. Using DW-MRI, this phenomenon enables the detection of successful tumor response to PDT within 7 hours posttreatment. This process was validated by direct, histological, immunohistochemical examinations and also by evaluation of serum prostate-specific antigen (PSA) levels that decreased significantly already 7 hours posttreatment. In vitro studies of multicellular cell spheroids confirmed a PDT-induced decrease in ADC, suggesting that lipid peroxidation (LPO) significantly contributes to ADC decline observed after PDT. These results demonstrate that TOOKAD-based PDT successfully eradicates prostate adenocarcinoma xenografts and suggests DW-MRI to be useful for the detection of early tumor response and treatment outcome in the clinical setting.
To elucidate the immune aspects of insulin-dependent diabetes mellitus (IDDM), we attempted to generate human monoclonal anti-insulin antibodies by fusing peripheral blood lymphocytes obtained from 10 insulin-treated IDDM patients with cells from a human lymphoblastoid cell line. Hybridomas that secreted immunoglobulins appeared in 9 of 400 wells. One of these hybridomas secreted anti-insulin antibody of the IgM class. The lymphocytic partner of this hybridoma was obtained from an IDDM patient who had undetectable levels of antibodies to insulin in his serum. Thus, by employing the hybridoma technique, it was possible to reveal the presence of insulin-sensitized B-lymphocytes in a patient who was serologically negative for anti-insulin antibodies. The monoclonal antibody recognized intact human insulin and insulins of other species, but not isolated A- and B-chains. This indicates that the antibody was functionally an autoantibody directed to an epitope formed by the native conformation of a highly conserved portion of the insulin molecule. This is the first report of a human hybridoma antibody to insulin.
Delayed-type hypersensitivity (DTH) responses served in this study as an experimental model for the analysis of genetic regulations of T-cell responses. Educated irradiated cells from H-2b mice mediated responses in syngeneic recipients, whereas mice of the a, d, f, k, and s haplotypes were nonresponders to poly(LTyr,LGlu)-poly(DLAla)--poly(LLys)[(T,G)-A--L]. These results suggest that cell-mediated immune responsiveness to (T,G)-A--L is linked to the H-2 complex, as was shown for humoral responses. Educated irradiated T cells of F1 hybrids between high and low responders mediated DTH responses, which indicates that the gene(s) controlling the DTH responses is dominant. To analyze the genetic defect in DTH responses to (T,G)-A--L, we separated the T-cell activation phase from the effector phase that was determined in recipient mice. Two types of nonresponders were observed: (a) When lymphocytes of the a or k haplotypes were educated in a syngeneic environment and then transferred into hybrids between the parental (nonresponder x responder) F1 recipients, DTH responses could have been manifested. (b) On the other hand, no DTH responses could be mediated by transferring educated cells of the H-2s or H-2f origin into the appropriate F1 recipients. In addition, irradiated F1 cells that had been activated to (T,G)-A--L could not mediate DTH responses in both types of nonresponder recipients. These results suggest that T cells of H-2k or H-2a mice can be activated to generate DTH responses to (T,G)-A--L and that the defect in these mouse strains is expressed in another cell population needed for the manifestation of the DTH reaction in the recipient mice. In contrast, T cells of H-2s and H-2f origin cannot be activated to (T,G)-A--L and, thus, fail to manifest DTH responses.
