RESUMO
Cellular and nuclear uptake of dual labelled conjugates could be of great value for chemotherapy and cancer diagnostics. Therefore we designed conjugates in which gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a contrast agent for magnetic resonance imaging and fluorescein isothiocyanate (FITC), a fluorescence marker were coupled to membrane translocation sequences (MTS). The MTSs we employed were the third helix of the Antennapedia homeodomain, the HIV-1 Tat peptide and the N-myristoylated HIV-1 Tat peptide. We used confocal laser scanning microscopy, fluorescence activated cell sorting, magnetic resonance imaging (MRI) and viability tests to examine the cellular and nuclear uptake of these conjugates into U373 glioma cells, as well as their cytotoxic effects. We found that the Antennapedia conjugate was taken up by no more than 20% of the cells. The HIV-1 Tat conjugate showed even lower uptake into less than 3% of cells. Interestingly, N-myristoylation of the HIV-1 Tat conjugate drastically improved its cellular uptake. Up to 70% of cells showed cellular and nuclear uptake of the N-myristoylated HIV-1 Tat conjugate. Conjugate cytotoxicity appears to correlate with cellular uptake.
Assuntos
Cátions/metabolismo , Compostos Heterocíclicos/metabolismo , Compostos Organometálicos/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Transporte Biológico/fisiologia , Cátions/química , Linhagem Celular Tumoral , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Compostos Heterocíclicos/química , Humanos , Dados de Sequência Molecular , Ácido Mirístico/metabolismo , Compostos Organometálicos/química , Peptídeos/química , Peptídeos/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Peptide conjugates derived from the SV 40 T antigen nuclear localisation sequence (NLS) have been successfully used to translocate both fluorescein isothiocyanate (FITC) and Gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) into the cytoplasm and nucleus of glioma cells. However, uptake occurred only in up to 35% of cells. To improve cellular uptake, we designed three novel FITC-labelled Gd-DOTA conjugates. In the first conjugate, the commonly used Gd-DOTA-complex was coupled to the nuclear localization sequence (NLS) of the Simian Virus (SV) 40 T antigen alone as a control. In the second conjugate, the Gd-DOTA-coupled SV 40 T antigen NLS was elongated by the HIV-1 tat peptide (HIV-NLS). A third conjugate, in which the Gd-DOTA-complex was coupled to the SV 40 T antigen NLS elongated by a peptide containing seven arginines and six aminohexanoic acids (Ahx6R7) was also synthesized (AHX-NLS). By means of confocal laser scanning microscopy, fluorescence activated cell sorting, magnetic resonance imaging (MRI) and viability tests we were able to demonstrate that the first conjugate containing only the NLS of the SV 40 T antigen stained the nuclei of no more than 10-12% of U373 and LN18 glioma cells, resulting in low signal intensity in MRI. The stained cells remained viable. After incubation with conjugates HIV-NLS and AHX-NLS the nuclei of up to 73% of U373 and LN18 glioma cells were stained. This was associated with high signal intensity in MRI and cell death. As previously shown, the gadolinium ion reduces cellular uptake of DOTA conjugates. To confirm this, the conjugates were produced with or without gadolinium. The gadolinium-free DOTA conjugates showed a higher cellular uptake rate and an increased cytotoxic potential.
Assuntos
Núcleo Celular/metabolismo , Corantes Fluorescentes/metabolismo , Compostos Heterocíclicos com 1 Anel/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Transporte Ativo do Núcleo Celular , Antígenos Transformantes de Poliomavirus/química , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/metabolismo , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes/química , Glioma/patologia , Compostos Heterocíclicos/metabolismo , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos com 1 Anel/farmacologia , Humanos , Imageamento por Ressonância Magnética , Microscopia Confocal , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Compostos Organometálicos/metabolismo , Compostos Organometálicos/farmacologia , Peptídeos/síntese química , Espectrometria de Massas por Ionização por Electrospray , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is commonly used as contrast agent in magnetic resonance imaging (MRI), but cannot enter the cytoplasm or cell nucleus. We designed a tetrapeptide carrying fluorescein isothiocyanate (FITC) and Gd-DOTA. This conjugate was coupled to the nuclear localisation sequence (NLS) of the Simian Virus (SV) 40 T antigen elongated by four arginines. In a second conjugate one lysine of the original SV 40 T antigen NLS was replaced by threonine. An FITC-labelled DOTA-tetrapeptide conjugate lacking the NLS peptide served as a negative control. We tried to achieve sequence specific entry of the Gd-DOTA-complex into the cytoplasm and nucleus of human U373 and LN18 glioma cells. Using confocal laser scanning microscopy (CLSM), fluorescence activated cell sorting (FACS), magnetic resonance imaging (MRI) and viability tests we found that both NLS conjugates stained the cell nuclei of U373 and LN18 glioma cells, represented also by a rise in signal intensity compared to the native control in MRI. The majority of stained cells remained viable. All conjugates were also produced without Gd. The Gd-free DOTA-conjugates showed an increase in cellular uptake rate. Conjugate cytotoxicity correlated closely to cellular uptake. Gd-containing DOTA-conjugates directed to the cytoplasm or the nucleus may be the basis for the development of novel diagnostic agents.
Assuntos
Meios de Contraste/metabolismo , Compostos Heterocíclicos com 1 Anel/metabolismo , Compostos Heterocíclicos/metabolismo , Sinais de Localização Nuclear/metabolismo , Compostos Organometálicos/metabolismo , Peptídeos/metabolismo , Linhagem Celular Tumoral , Núcleo Celular , Meios de Contraste/química , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/metabolismo , Compostos Heterocíclicos/química , Compostos Heterocíclicos com 1 Anel/química , Humanos , Imageamento por Ressonância Magnética , Microscopia Confocal , Sinais de Localização Nuclear/química , Compostos Organometálicos/química , Peptídeos/síntese química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The seven N-terminal amino acids AVPIAQK (SmacN7) of the mitochondrial protein Smac (second mitochondria-derived activator of caspase) promote caspase activation by binding specifically to inhibitor of apoptosis proteins (IAPs) and blocking their inhibitory activity. SmacN7 cannot pass through the cell membrane, but to be of therapeutic use it would be essential for it to enter the cell. To achieve transmembrane transport of SmacN7 we coupled it to a novel fluorescein isothiocyanate (FITC)-labelled transmembrane transport peptide RRRRK(FITC)RRRR via ss-alanine to produce the conjugate AVPIAQKssA RRRRK(FITC)RRRR. Because IAPs are much more strongly expressed in the cytoplasm of tumor cells, we expected this conjugate to produce staining of the cytoplasm, and for this to be stronger in tumor cells than in healthy cells. Surprisingly, we found strong nuclear uptake of the Smac conjugate and of the transport peptide alone without subsequent release in both tumor cells and healthy cells from the bladder, prostate, and brain. This was accompanied by cell death. In contrast to expectations, it appears that the apoptotic effects observed do not result from the SmacN7 cargo alone.
Assuntos
Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Proteínas Mitocondriais/síntese química , Proteínas Mitocondriais/farmacologia , Peptídeos/química , Neoplasias da Próstata/patologia , Sequência de Aminoácidos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Saúde , Humanos , Masculino , Proteínas Mitocondriais/químicaRESUMO
Long slender trypanosomes, isolated from infected mouse blood or from cryopreserved stabilates, respectively, were unable to grow in conditioned media (cMEM), prepared from the declining phase of axenic bloodstream form cultures. Additionally, mixtures of fresh medium and cMEM led to decreased growth rates and, in accordance to the amount of cMEM used, to a decreased S-adenosyl-L-methionine decarboxylase (Ado-MetDC; E.C. 4.1.1.50) activity. Since addition of polyamines could not overcome the process of transition from dividing to non-dividing cells and the intracellular S-adenosyl-L-methionine (AdoMet), ornithine and putrescine concentrations seemed unaltered during the course of cultivation, we questioned if polyamine metabolism is involved in this transition process. Activities of two key enzymes of polyamine metabolism, AdoMetDC and ornithine decarboxylase (ODC; E.C. 4.1.1.17) were therefore monitored during different growth stages. Our results revealed a specific activity of 44 pmol min-1 mg protein-1 for AdoMetDC and a KM of 10 mu M for AdoMet. Methylglyoxal bis(guanylhydrazone) showed a Ki of 6 mu M. The constant activity of the enzyme during a 7 h time-course in the presence of cycloheximide indicates a t1/2 of more than 7 h for the trypanosomal enzyme. Enzyme activity in trypanosomes isolated from infected laboratory animals and from logarithmic phase bloodstream or procyclic form cultures was high according to a high dividing rate, whereas enzyme activity in parasites isolated from the stationary phase of bloodstream from culture was negligible. In these cultures, AdoMetDC activity decreased with a t1/2 of 7 h during transition from long slender to short stumpy-like forms as soon as the stationary phase was reached. ODC activity was high (approximately 300 pmol min-1 mg protein-1) in dividing trypanosomes isolated from infected animals as well as from logarithmic phase bloodstream or procyclic form cultures and decreased also during transition with a t1/2 of 10 h.
Assuntos
Adenosilmetionina Descarboxilase/genética , Vida Livre de Germes , Biossíntese de Proteínas , Trypanosoma brucei brucei/genética , Animais , Diferenciação Celular/fisiologia , Técnicas de Cultura , Regulação para Baixo , Modelos Logísticos , Ornitina Descarboxilase/metabolismo , Trypanosoma brucei brucei/enzimologiaRESUMO
We have recently described a method for identifying contact sites between actin and thymosin beta4 (Tbeta4) by following spectrophotometrically the extent and kinetics of distinct, thiol-specific crosslinking reactions between appropriate derivatives of the two proteins [Reichert et a]. (1996) J. Biol. Chem. 271, 1301-1308]. In the present study this method was used to show that such crosslinking, which is indicative of complex formation, occurs to the same extent with the actin-DNase I complex as with pure actin, although at a somewhat lower rate. Further evidence for the formation of the ternary complex was given by gel electrophoresis. From fluorescence spectroscopy the KD value of Tbeta4 from the actin-DNase I complex was found to be identical to that from pure actin. In line with these data, the capacity of actin for inhibiting DNase I was not affected by the addition of Tbeta4. In conclusion, DNase I and Tbeta4 are independent of each other in their interaction with actin, suggesting that the binding sites of thymosin beta4 and DNase I on actin do not overlap. A ternary complex of DNase I, actin and Tbeta4, if obtained in crystalline form, could thus provide an approach for studying the interface of Tbeta4 and actin by X-ray analysis.
Assuntos
Actinas/metabolismo , Desoxirribonuclease I/metabolismo , Timosina/metabolismo , Animais , Bovinos , Reagentes de Ligações Cruzadas , Desoxirribonuclease I/antagonistas & inibidores , Estrutura Molecular , Coelhos , Espectrometria de FluorescênciaRESUMO
Two rabbit polyclonal antisera, one directed against thymosin beta 4 and the other one against the peptide fragment thymosin beta 4 (1-14) were characterised by epitope mapping. Hexapeptides representing the whole sequence of the native peptide and overlapping by one amino acid were synthesised on polystyrene pins. The antigenic determinants were identified in microtitre plates with an ELISA procedure. The polyclonal serum against thymosin beta 4 detected three epitopes (N-terminal, mid-region and C-terminal) whereas the polyclonal serum against the fragment contained only antibodies specific for the N-terminal epitope. These experimental results are consistent with theoretical predictions.
Assuntos
Timosina/imunologia , Sequência de Aminoácidos , Mapeamento de Epitopos , Dados de Sequência Molecular , Oligopeptídeos/imunologiaRESUMO
Cross-inhibition by quorum-sensing pheromones between Staphylococcus aureus and Staphylococcus epidermidis was investigated using all known S. aureus agr pheromone subgroups. All S. aureus subgroups were sensitive towards the S. epidermidis pheromone, with the exception of the recently identified subgroup 4. The subgroup 4 pheromone was also the only S. aureus pheromone able to inhibit the S. epidermidis agr response. The close relation of subgroup 4 to subgroup 1 suggests that subgroup 4 might have evolved from subgroup 1 by mutation under the selective pressure of competition with S. epidermidis. The competition between S. aureus and S. epidermidis by means of quorum-sensing cross talk seems to be generally in favor of S. epidermidis, which might explain the predominance of S. epidermidis on the skin and in infections on indwelling medical devices.
Assuntos
Feromônios/farmacologia , Transdução de Sinais , Staphylococcus aureus/fisiologia , Staphylococcus epidermidis/fisiologia , Transativadores , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/biossíntese , Evolução Molecular , Testes de Sensibilidade Microbiana , Feromônios/classificação , Staphylococcus aureus/citologia , Staphylococcus epidermidis/citologia , Fatores de Transcrição/metabolismoRESUMO
Comparison of high performance liquid chromatograms (HPLC) with capillary electrophoresis shows the latter to be superior in many cases owing to rapid separation, high resolution and high sensitivity. This is demonstrated with two examples (i) the isolation of natural peptides from bovine tissue and (ii) characterization of a synthetic peptide mixture with the natural sequence (fragment) of the human immunodeficiency virus (HIV) transmembrane glycoprotein gp 41 env.
Assuntos
Eletroforese/métodos , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/métodos , Estudos de Avaliação como Assunto , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/isolamento & purificação , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos/química , Timosina/química , Timosina/isolamento & purificaçãoRESUMO
A new radioimmunoassay has been developed for thymosin beta 4 by generating rabbit polyclonal antibodies against the synthetic N-terminal peptide fragment 1-15 coupled to KLH. The synthetic analogue [Tyr12]-thymosin beta 4 (1-15) was used as tracer. This radioimmunoassay, with a useful range of 10-1000 pmoles, showed cross-reactivity with the second homologous beta-thymosin of man and rat (thymosin beta 10) but not of calf (thymosin beta 9). This radioimmunoassay, together with an improved radioimmunoassay for the N-terminus of parathymosin alpha, was employed for the measurement of the levels of thymosin beta 4 and parathymosin alpha in nuclear and extranuclear extracts of calf thymus. The bulk of these polypeptides was found in the extranuclear material whereas only traces were observed in the nuclear environment, which indicates the extranuclear localisation of alpha- and beta-thymosins.
Assuntos
Timosina/análise , Timo/química , Animais , Bovinos , Núcleo Celular/química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Radioimunoensaio/métodos , Frações Subcelulares/químicaRESUMO
Binding sites of actin and thymosin beta 4 were investigated using a set of bifunctional thiol-specific reagents, which allowed the insertion of cross-linkers of defined lengths between cysteine residues of the complexed proteins. After the cross-linkers were attached to actin specifically at either Cys10, Cys374, or the sulfur atom of the ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S), the actin derivatives were reacted with synthetic thymosin beta 4 analogs containing a cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate cross-linking as followed by UV spectroscopy was found for Cys374 of actin and Cys6 of thymosin beta 4, indicating that the N terminus of thymosin beta 4 is in close proximity (< or = 9.2 A) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin beta 4 analogs when the cross-linkers were anchored at Cys10 of actin. A second contact site was identified by cross-linking of Cys17 and Cys28 in thymosin beta 4 with the ATP gamma S derivative bound to actin, indicating that the hexamotif of thymosin beta 4 (positions 17-22) is in close proximity (< or = 9.2 A) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin beta 4 for the interaction with actin was emphasized by the finding that thymosin analogs containing cysteine in these positions exhibited strongly reduced abilities to inhibit actin polymerization.
Assuntos
Actinas/química , Actinas/metabolismo , Timosina/química , Timosina/metabolismo , Actinas/isolamento & purificação , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Reagentes de Ligações Cruzadas , Cistina , Eletroforese em Gel de Poliacrilamida , Pulmão/metabolismo , Matemática , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Modelos Estruturais , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Peptídeos/síntese química , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Coelhos , Relação Estrutura-Atividade , Compostos de Sulfidrila , Timosina/isolamento & purificaçãoRESUMO
Solid-phase synthesis of triple-helical peptides, including native collagen III sequences, was started with a trimeric branch, based upon the lysine dipeptide [Fields, C. G., Mickelson, D. J., Drake, S. L., McCarthy, J. B., and Fields, G. B. (1993) J. Biol. Chem. 268, 14153-14160]. Branch synthesis was modified, using TentaGel R as resin, p-hydroxybenzyl alcohol (HMP) as linker, Dde as N(epsilon)-protective group, and HATU/HOAT as coupling reagent. Three homotrimeric sequences, each containing the Gly 606-Gly 618 portion of human type III collagen, were added to the amino functions of the branch. The final incorporation of GlyProHyp triplets, stabilizing the collagen III triple helix, was performed by using protected GlyProHyp tripeptides, each containing tert-butylated hydroxyproline [P(tBu)] instead of hydroxyproline (P). Among the protected tripeptides FmocP(tBu)PG, FmocPP(tBu)G, and FmocGPP(tBu), prepared manually on a chlorotrityl resin, incorporation of FmocPP(tBu)Gly was best suited for synthesis of large and stable peptides, such as PPG(8), containing 8 (PPG)(3) trimers (115 residues, 10 610 Da). The structures of five peptides, differing from each other by the type and number of the triplets incorporated, were verified by MALDI-TOF-MS. Their conformations and thermodynamic data were studied by circular dichroism and differential scanning calorimetry. Except for PPG(8), containing 8 (PPG)(3) trimers with hydroxyproline in the X position and adopting a polyproline II structure, all peptides were triple-helical in 0.1 M acetic acid and their thermal stabilities ranged from t(1/2) = 39. 4 to t(1/2) = 62.5 degrees C, depending on the identity and number of the triplets used. Similar values of the van't Hoff enthalpy, DeltaH(vH), derived from melting curves, and the calorimetric enthalpy, DeltaH(cal), obtained from heat capacity curves, indicate a cooperative ratio of CR = DeltaH(vH)/DeltaH(cal) = 1, establishing a two-state process for unfolding of THP(III) peptides. The independence of the transition temperatures t(1/2) on peptide concentration as well as equilibrium centrifugation data indicate monomolecular dimer(f) to dimer(u) (F(2) <--> U(2)) transitions and, in addition, bimolecular dimer(f) to monomer(u) transitions (F(2) <--> 2U). The dominance of the concentration-independent monomolecular reaction over the concentration-dependent bimolecular reaction makes thermal unfolding of THP(III) peptides appear to be monomolecular. If one designates the molecularity described by the term pseudomonomolecular, unfolding of the dimeric peptides PPG(6-8) follows a two-state, pseudomonomolecular reaction.
Assuntos
Colágeno/síntese química , Peptídeos/síntese química , Dobramento de Proteína , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Colágeno/isolamento & purificação , Glicina/química , Hidroxiprolina/química , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/isolamento & purificação , Peptídeos/isolamento & purificação , Prolina/química , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Termodinâmica , UltracentrifugaçãoRESUMO
HER-2/neu oncoprotein contains several major histocompatibility complex class I-restricted epitopes, which are recognised by cytotoxic T lymphocyte (CTL) on autologous tumours and therefore can be used in immune-based cancer therapies. Of these, the most extensively studied is HER-2(9(369)). In the present report, we used dendritic cells pulsed with HER-2(9(369)) to stimulate, in the presence of IL-7 and IL-12, the production of IFN-gamma by patients' CTL detected by the enzyme-linked immunosorbent spot-assay. Frequencies of peptide-specific precursors were estimated in HLA-A2, HLA-A3 and HLA-A26 patients with HER-2/neu-positive (+) breast, ovarian, lung, colorectal and prostate cancers and healthy individuals. We found increased percentages of such precursors in HLA-A2 (25%) and HLA-A26 (30%) patients, which were significantly higher (60%) in HLA-A3 patients. Our results demonstrate for the first time that pre-existing immunity to HER-2(9(369)) occurs in patients with colorectal, lung and prostate cancer. They also suggest that HER-2(9(369)) can be recognised by CTL, besides HLA-A2, also in the context of HLA-A3 and HLA-A26, thus increasing the applicability of HER-2(9(369))-based vaccinations in a considerably broader patients' population.
Assuntos
Células Dendríticas/imunologia , Neoplasias/imunologia , Fragmentos de Peptídeos/imunologia , Receptor ErbB-2/metabolismo , Linfócitos T Citotóxicos/imunologia , Adulto , Idoso , Divisão Celular , Citocinas/metabolismo , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Feminino , Antígenos HLA-A/imunologia , Antígeno HLA-A2/imunologia , Antígeno HLA-A3/imunologia , Humanos , Imunização , Imunofenotipagem , Interferon gama/biossíntese , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
HER2/neu-derived peptides inducing MHC class II-restricted CD4+ T helper lymphocyte (Th) responses, although critical for tumour rejection, are not thoroughly characterized. Here, we report the generation and characterization of CD4+ T cell clones specifically recognizing a HER-2/neu-derived peptide (776-788) [designated HER2(776-788)]. Such clones yielded specific proliferative and cytokine [gamma-interferon(IFN)-gamma] responses when challenged with autologous dendritic cells (DCs) loaded with HER2(776-788). By performing blocking studies with monoclonal antibodies (MAbs) and by using DCs from allogeneic donors sharing certain HLA-DR alleles, we found that HER2(776-788) is a promiscuous peptide presented, at least, by DRB5*0101, DRB1*0701 and DRB1*0405 alleles. One TCRV beta 6.7+ clone recognized the HLA-DRB5*0101+ FM3 melanoma cell line transfected with a full length HER-2/neu cDNA. Moreover, this clone recognized the HER-2/neu+ SKBR3 breast cancer cell line induced to express HLA-DR, thus demonstrating that HER2(776-788) represents a naturally processed and presented epitope. Our data demonstrate that helper peptide HER2(776-788) represents a promiscuous epitope binding to at least three HLA-DR alleles, thus offering a broad population coverage. The use of antigenic peptides presented by major histocompatibility complex (MHC) class II in addition to those presented by class I may improve the therapeutic efficacy of active immunization.