DNA vaccination against the novel tumor antigen, MUC18, for the therapy of metastatic melanoma

Melanoma patients with metastases have a very low survival rate and limited treatment options. Therefore, the targeting of melanoma cells when they begin to invade and metastasize would be beneficial. A specific adhesion molecule that is upregulated at the vertical growth phase is the melanoma cell adhesion molecule (MCAM/MUC18). MUC18 is expressed in late primary and metastatic melanoma with little or no expression on normal melanocytes. MUC18 has been demonstrated to have a role in the progression and metastasis of human melanoma. We utilized the alphavirus-based DNA plasmid, SINCp, encoding full length human MUC18 for vaccination against B16F10 murine melanoma cells expressing human MUC18. The alphavirus-based DNA plasmid leads to the expression of large quantities of heterologous protein as well as danger signals due to dsRNA intermediates produced during viral replication. In a preventative primary tumor model and an experimental tumor model, mice vaccinated against human MUC18 had decreased tumor incidence and reduced lung metastases when challenged with B16F10 murine melanoma cells expressing human MUC18. In a therapeutic tumor model, vaccination against human MUC18 reduced the tumor burden in mice with pre-existing lung metastases but did not have a significant effect on therapeutic vaccination in a primary tumor model. We next cloned murine MUC18 into SINCp for use in determining the efficacy of vaccination against murine MUC18 in a syngeneic animal model. Mice were vaccinated and challenged in a primary tumor and experimental metastasis model. In both models, vaccination significantly reduced tumor incidence and lung metastases. Humoral and cell-mediated responses were then determined. Flow cytometry and immunohistochemistry showed that specific antibodies were developed from vaccination against both human and murine MUC18. IgG2a antibody isotype was also developed indicating a Th1 type response. ELISPOT results showed that mice vaccinated against human MUC18 created a specific T cell response to targets expressing human MUC18. Mice vaccinated against murine MUC18 raised specific effector cells against target cells expressing murine MUC18 in a cell killing assay. These results indicate that vaccination against MUC18 developed specific immune responses against MUC18 and were effective in controlling tumor growth in melanoma expressing MUC18. ^

IL -12 inhibits the metastatic potential of osteosarcoma cells and induces tumor regression via a mechanism involving Fas /Fas ligand pathway

Despite multiple changes in the adjuvant chemotherapy regimens used to treat osteosarcoma (OS), the 2-year metastasis-free survival has remained at 65–70% for the past 10 years. Characterizing the molecular determinants that permit metastatic spread of tumor cells is a crucial element in developing new approaches for the treatment of osteosarcoma. Since OS metastasizes almost exclusively to the lung, an organ with constitutive Fas ligand (FasL) expression, we hypothesized that the expression of Fas (CD95, APO-1) by OS cells may play a role in the ability of these cells to form lung metastases. Fas expression was quantified in human SAOS-2 OS cells and selected variants (LM2, LM4, LM5, LM6, LM7). Using northern blot, FACS and RT-PCR analysis, low Fas expression was found to correlate with higher metastatic potential in these cell lines. The highly metastatic LM7 cell line was transfected with the full-length human Fas gene and injected into athymic nude mice. The median number of metastatic nodules per mouse fell from over 200 to 1.1 and the size of the nodules decreased from a range of 0.5–9.0 mm to less than 0.5 mm in the Fas-transfected cell line compared to the native LM7 cell line. Additionally, the subsequent incidence of lung metastases was lower in the Fas-expressing cell line. IL-12 was seen to upregulate Fas expression in the highly metastatic LM sublines in vitro. To visualize the effects of IL-12 in vivo, nude mice were injected with LM7 cells and treated biweekly for 4 weeks with Ad.mIL-12, saline control or Ad.βgal. Lung sections were analyzed via immunchistochemistry for Fas expression. A higher expression of Fas was found in tumors from mice receiving IL-12. To study the mechanism by which IL-12 upregulates Fas, LM7 cells were transfected with a luciferase reporter gene construct containing the full-length human fas promoter. Treatment with IL-12 increased luciferase activity. We therefore conclude that IL-12 influences the metastatic potential of OS cells by upregulating the fas promoter, resulting in increased cell surface Fas expression and susceptibility to Fas-induced cell death. ^

p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting

DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA. By using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full-length human wild-type (wt) p53. Our data show that four subunits of p53DBD bend the DNA by 32–36°, whereas wt p53 bends it by 51–57°. The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. More sophisticated phasing analyses also demonstrate that p53DBD and wt p53 overtwist the DNA response element by ≈35° and ≈70°, respectively. These results are in accord with molecular modeling studies of the tetrameric complex. Within the constraints imposed by the protein subunits, the DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53–DNA tetrameric complex is stabilized by DNA overtwisting and bending toward the major groove at the CATG tetramers. This bending is consistent with the inherent sequence-dependent anisotropy of the duplex. Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous interprotein interactions that increase the stability and cooperativity of binding. The novel architecture of the p53 tetrameric complex has important functional implications including possible p53 interactions with chromatin.

Anti-β2 glycoprotein I (β2GPI) autoantibodies recognize an epitope on the first domain of β2GPI

Anticardiolipin (aCL) autoantibodies are associated with thrombosis, recurrent fetal loss, and thrombocytopenia. Only aCL found in autoimmune disease require the participation of the phospholipid binding plasma protein β2 glycoprotein I (β2GPI) for antibody binding and now are called anti-β2GPI. The antigenic specificity of aCL affinity purified from 11 patients with high titers was evaluated in an effort to better understand the pathophysiology associated with aCL. Seven different recombinant domain-deleted mutants of human β2GPI, and full length human β2GPI (wild-type), were used in competition assays to inhibit the autoantibodies from binding to immobilized wild-type β2GPI. Only those domain-deleted mutants that contained domain 1 inhibited the binding to immobilized wild-type β2GPI from all of the patients. The domain-deleted mutants that contained domain 1 inhibited all aCL in a similar but not identical pattern, suggesting that these aCL recognize a similar, but distinguishable, epitope(s) present on domain 1.

Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.

Mechanisms of Human Mitochondrial DNA Maintenance: The Determining Role of Primary Sequence and Length over Function

Although the regulation of mitochondrial DNA (mtDNA) copy number is performed by nuclear-coded factors, very little is known about the mechanisms controlling this process. We attempted to introduce nonhuman ape mtDNA into human cells harboring either no mtDNA or mutated mtDNAs (partial deletion and tRNA gene point mutation). Unexpectedly, only cells containing no mtDNA could be repopulated with nonhuman ape mtDNA. Cells containing a defective human mtDNA did not incorporate or maintain ape mtDNA and therefore died under selection for oxidative phosphorylation function. On the other hand, foreign human mtDNA was readily incorporated and maintained in these cells. The suicidal preference for self-mtDNA showed that functional parameters associated with oxidative phosphorylation are less relevant to mtDNA maintenance and copy number control than recognition of mtDNA self-determinants. Non–self-mtDNA could not be maintained into cells with mtDNA even if no selection for oxidative phosphorylation was applied. The repopulation kinetics of several mtDNA forms after severe depletion by ethidium bromide treatment showed that replication and maintenance of mtDNA in human cells are highly dependent on molecular features, because partially deleted mtDNA molecules repopulated cells significantly faster than full-length mtDNA. Taken together, our results suggest that mtDNA copy number may be controlled by competition for limiting levels of trans-acting factors that recognize primarily mtDNA molecular features. In agreement with this hypothesis, marked variations in mtDNA levels did not affect the transcription of nuclear-coded factors involved in mtDNA replication.

Compilation of somatic mutations of the CDKN2 gene in human cancers : non-random distribution of base substitutions

The CDKN2 gene, encoding the cyclin-dependent kinase inhibitor p16, is a tumour suppressor gene that maps to chromosome band 9p21-p22. The most common mechanism of inactivation of this gene in human cancers is through homozygous deletion; however, in a smaller proportion of tumours and tumour cell lines intragenic mutations occur. In this study we have compiled a database of over 120 published point mutations in the CDKN2 gene from a wide variety of tumour types. A further 50 deletions, insertions, and splice mutations in CDKN2 have also been compiled. Furthermore, we have standardised the numbering of all mutations according to the full-length 156 amino acid form of p16. From this study we are able to define several hot spots, some of which occur at conserved residues within the ankyrin domains of p16. While many of the hotspots are shared by a number of cancers, the relative importance of each position varies, possibly reflecting the role of different carcinogens in the development of certain tumours. As reported previously, the mutational spectrum of CDKN2 in melanomas differs from that of internal malignancies and supports the involvement of UV in melanoma tumorigenesis. Notably, 52% of all substitutions in melanoma-derived samples occurred at just six nucleotide positions. Nonsense mutations comprise a comparatively high proportion of mutations present in the CDKN2 gene, and possible explanations for this are discussed.

Selective cleavage of human sex hormone-binding globulin by kallikrein-related peptidases and effects on androgen action in LNCaP prostate cancer cells

Stimulation of the androgen receptor via bioavailable androgens, including testosterone and testosterone metabolites, is a key driver of prostate development and the early stages of prostate cancer. Androgens are hydrophobic and as such require carrier proteins, including sex hormone-binding globulin (SHBG), to enable efficient distribution from sites of biosynthesis to target tissues. The similarly hydrophobic corticosteroids also require a carrier protein whose affinity for steroid is modulated by proteolysis. However, proteolytic mechanisms regulating the SHBG/androgen complex have not been reported. Here, we show that the cancer-associated serine proteases, kallikrein-related peptidase (KLK)4 and KLK14, bind strongly to SHBG in glutathione S-transferase interaction analyses. Further, we demonstrate that active KLK4 and KLK14 cleave human SHBG at unique sites and in an androgen-dependent manner. KLK4 separated androgen-free SHBG into its two laminin G-like (LG) domains that were subsequently proteolytically stable even after prolonged digestion, whereas a catalytically equivalent amount of KLK14 reduced SHBG to small peptide fragments over the same period. Conversely, proteolysis of 5α-dihydrotestosterone (DHT)-bound SHBG was similar for both KLKs and left the steroid binding LG4 domain intact. Characterization of this proteolysis fragment by [(3)H]-labeled DHT binding assays revealed that it retained identical affinity for androgen compared with full-length SHBG (dissociation constant = 1.92 nM). Consistent with this, both full-length SHBG and SHBG-LG4 significantly increased DHT-mediated transcriptional activity of the androgen receptor compared with DHT delivered without carrier protein. Collectively, these data provide the first evidence that SHBG is a target for proteolysis and demonstrate that a stable fragment derived from proteolysis of steroid-bound SHBG retains binding function in vitro.

Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice

We have investigated the role of bone sialoprotein (BSP), a secreted glycoprotein normally found in bone, in breast cancer progression. To explore functions for BSP in human breast cancer invasion and metastasis, the full-length BSP cDNA was transfected into the MDA-MB-231-BAG human breast cancer cell line under the control of the CMV promoter. Clones expressing BSP and vector control clones were isolated. BSP producing clones showed increased monolayer wound healing, a faster rate of stellate outgrowth in Matrigel and increased rate of invasion into a collagen matrix when compared to control clones. Clones were also examined in models of breast cancer growth and metastasis in vivo. BSP transfected clones showed an increased rate of primary tumor growth following mammary fat pad injection of nude mice. BSP transfected clones and vector control clones metastasized to soft organs and bone at a similar rate after intra-cardiac injection as determined by real-time PCR and X-ray analysis. Although these organs were targets for both BSP transfected and non-transfected cells, the size of the metastatic lesion was shown to be significantly larger for BSP expressing clones. This was determined by real-time PCR analysis for soft organs and by X-ray analysis of bone lesions. For bone this was confirmed by intra-tibial injections of cells in nude mice. We conclude that BSP acts to drive primary and secondary tumor growth of breast cancers in vivo.

Human Torque teno virus: epidemiology, cell biology and immunology

Torque teno virus (TTV) was discovered in 1997 in the serum of a Japanese patient who had a post-transfusion hepatitis of unknown etiology. It is a small virus containing a circular single-stranded DNA genome which is unique among human viruses. Within a few years after its discovery, the TTVs were noted to form a large family of viruses with numerous genotypes. TTV is highly prevalent among the general population throughout the world, and persistent infections and co-infections with several genotypes occur frequently. However, the pathogenicity and the mechanism for the sustained occurrence of the virus in blood are at present unclear. To determine the prevalence of TTV in Finland, we set up PCR methods and examined the sera of asymptomatic subjects for the presence of TTV DNA and for genotype-6 DNA. TTV was found to be highly prevalent also in Finland; 85% of adults harbored TTV in their blood, and 4% were infected with genotype-6. In addition, TTV DNA was detected in a number of different tissues, with no tissue-type or symptom specificity. Most cell-biological events during TTV infections are at the moment unknown. Replicating TTV DNA has, however, been detected in liver and the hematopoietic compartment, and three mRNAs are known to be generated. To characterize TTV cell biology in more detail, we cloned in full length the genome of TTV genotype 6. We showed that in human kidney-derived cells TTV produces altogether six proteins with distinct subcellular localizations. TTV mRNA transcription was detected in all cell lines transfected with the full-length clone, and TTV DNA replicated in several of them, including those of erythroid, kidney, and hepatic origin. Furthermore, the viral DNA replication was shown to utilize the cellular DNA polymerases. Diagnoses of TTV infections have been based almost solely on PCR, whereas serological tests, measuring antibody responses, would give more information on many aspects of these infections. To investigate the TTV immunology in more detail, we produced all six TTV proteins for use as antigens in serological tests. We detected in human sera IgM and IgG antibodies to occur simultaneously with TTV DNA, and observed appearance of TTV DNA regardless of pre-existing antibodies, and disappearance of TTV DNA after antibody appearance. The genotype-6 nucleotide sequence remained stable for years within the infected subjects, suggesting that some mechanism other than mutations is used by this minute virus to evade our immune system and to establish chronic infections in immunocompetent subjects.

Human parvovirus B19: tissue persistence and prevalence of prototypic and new variants

Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn2+, at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent crosslinking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.

High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (ICFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K-D similar to 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access In recent years, IGFBPs have been implicated in a variety of cancers However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Eschericha coli Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E coli and first structural characterization of a full-length IGFBP (C) 2010 Elsevier Inc. All rights reserved

Interplay between NS3 protease and human La protein regulates translation-replication switch of Hepatitis C virus

HCV NS3 protein plays a central role in viral polyprotein processing and RNA replication. We demonstrate that the NS3 protease (NS3(pro)) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding. More importantly, NS3(pro) binding to the SLIV hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, resulting in inhibition of HCV-IRES activity. Although overexpression of both NS3(pro) as well as the full length NS3 protein decreased the level of HCV IRES mediated translation, replication of HCV replicon RNA was enhanced significantly. These observations suggest that the NS3(pro) binding to HCV IRES reduces translation in favor of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might regulate the molecular switch from translation to replication of HCV.

Antibodies against the extracellular domain of human Notch1 receptor reveal the critical role of epidermal-growth-factor-like repeats 25-26 in ligand binding and receptor activation

The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs EGF (epidermal growth factor)-like-repeats] 11-12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21-30 and the ELRs 11-15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21-30 and ELRs 11-15 is similar to 10-fold stronger than that between ELRs 11-15 and the ligands. Although there was no interaction between Notch 1 ELRs 21-30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21-30 and 11-15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21-30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25-26 being the most effective. These results suggest that the ELRs 25-26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11-12 are the principal ligand-binding site, the ELRs 25-26 serve as a secondary binding site and play an important role in receptor activation.