Coxiella burnetii Infections in Small Ruminants and Humans in Switzerland.

The recent Q fever epidemic in the Netherlands raised concerns about the potential risk of outbreaks in other European countries. In Switzerland, the prevalence of Q fever in animals and humans has not been studied in recent years. In this study, we describe the current situation with respect to Coxiella (C.) burnetii infections in small ruminants and humans in Switzerland, as a basis for future epidemiological investigations and public health risk assessments. Specific objectives of this cross-sectional study were to (i) estimate the seroprevalence of C. burnetii in sheep and goats, (ii) quantify the amount of bacteria shed during abortion and (iii) analyse temporal trends in human C. burnetii infections. The seroprevalence of C. burnetii in small ruminants was determined by commercial ELISA from a representative sample of 100 sheep flocks and 72 goat herds. Herd-level seroprevalence was 5.0% (95% CI: 1.6-11.3) for sheep and 11.1% (95% CI: 4.9-20.7) for goats. Animal-level seroprevalence was 1.8% (95% CI: 0.8-3.4) for sheep and 3.4% (95% CI: 1.7-6) for goats. The quantification of C. burnetii in 97 ovine and caprine abortion samples by real-time PCR indicated shedding of >104 bacteria/g in 13.4% of all samples tested. To our knowledge, this is the first study reporting C. burnetii quantities in a large number of small ruminant abortion samples. Annual human Q fever serology data were provided by five major Swiss laboratories. Overall, seroprevalence in humans ranged between 1.7% and 3.5% from 2007 to 2011, and no temporal trends were observed. Interestingly, the two laboratories with significantly higher seroprevalences are located in the regions with the largest goat populations as well as, for one laboratory, with the highest livestock density in Switzerland. However, a direct link between animal and human infection data could not be established in this study.

Regulation of tamoxifen sensitivity by a PAK1-EBP1 signalling pathway in breast cancer.

BACKGROUND: EBP1, an ErbB3-binding protein, sensitises breast cancer cells to tamoxifen in part by decreasing ErbB2 protein levels. The p21-regulated serine/threonine kinase PAK1, implicated in tamoxifen resistance, phosphorylates EBP1 in vitro and in vivo at T261. Phosphorylation of EBP1 at this site induces tamoxifen resistance. We thus postulated that inhibition of PAK1 activity, by restoring EBP1 function, could ameliorate the hormone refractory phenotype of ErbB2-overexpressing breast cancer cells. METHODS: Effects of EBP1 on ErbB2 levels were measured by western blotting. Effects of EBP1 and IPA-3 on tamoxifen sensitivity were measured using a tetrazolium based cell viability assay. RESULTS: Transient transfection studies indicated that an EBP1 T261E mutant, which mimics EPB1 phosphorylated by PAK1, increased ErbB2 protein levels. An EBP1 T261A mutant, unable to be phosphorylated by PAK1, ameliorated PAK1-induced tamoxifen resistance, suggesting that phosphorylation of EBP1 by PAK1 contributes to tamoxifen resistance. We then tested if pharmacological inhibition of PAK1 activity might render hormone resistant cells, which endogenously overexpress PAK1, tamoxifen sensitive. IPA-3, a specific small MW PAK1 inhibitor, sensitised cells to tamoxifen only when EBP1 was ectopically expressed. IPA had no effect on tamoxifen resistance in T47D cells in which EBP1 protein had been ablated by shRNA. The IPA-induced increase in tamoxifen sensitivity was accompanied by a decrease in ErbB2 levels only in EBP1-overexpressing cells. CONCLUSION: These studies suggest that phosphorylation of EBP1 may be one mechanism of PAK1-induced hormone resistance and that PAK1 inhibitors may be useful in cells in which EBP1 is overexpressed.

Monoclonal antibodies to B and T lymphocyte attenuator (BTLA) have no effect on in vitro B cell proliferation and act to inhibit in vitro T cell proliferation when presented in a cis, but not trans, format relative to the activating stimulus.

B and T lymphocyte attenuator (BTLA) is an immunoglobulin superfamily member surface protein expressed on B and T cells. Its ligand, herpesvirus entry mediator (HVEM), is believed to act as a monomeric agonist that signals via the CRD1 of HVEM to inhibit lymphocyte activation: HVEM is also the receptor for lymphotoxin-α and LIGHT, which both bind in the CRD2 and CRD3 domains of the HVEM molecule, and for CD160 which competes with BTLA. We have shown that recombinant HVEM and a panel of different monoclonal antibodies specifically bind murine BTLA on both B and T cells and that some antibodies inhibit anti-CD3ε-induced T cell proliferation in vitro, but only when constrained appropriately with a putatively cross-linking reagent. The antibodies had no significant effect on in vitro T cell proliferation in a mixed lymphocyte reaction (MLR) assay nor on in vitro DO11.10 antigen-induced T cell proliferation. None of these antibodies, nor HVEM-Fc, had any significant effect on in vitro B cell proliferation induced by anti-immunoglobulin M antibodies (±anti-CD40) or lipopolysaccharide. We further elucidated the requirements for inhibition of in vitro T cell proliferation using a beads-based system to demonstrate that the antibodies that inhibited T cell proliferation in vitro were required to be presented to the T cell in a cis, and not trans, format relative to the anti-CD3ε stimulus. We also found that antibodies that inhibited T cell proliferation in vitro had no significant effect on the antibody captured interleukin-2 associated with the in vivo activation of DO11.10 T cells transferred to syngeneic recipient BALB/c mice. These data suggest that there may be specific structural requirements for the BTLA molecule to exert its effect on lymphocyte activation and proliferation.

Side-population cells in luminal-type breast cancer have tumour-initiating cell properties, and are regulated by HER2 expression and signalling.

BACKGROUND: The expression of side-population (SP) cells and their relation to tumour-initiating cells (T-ICs) have been insufficiently studied in breast cancer (BC). We therefore evaluated primary cell cultures derived from patients and a panel of human BC cell lines with luminal- or basal-molecular signatures for the presence of SP and BC stem cell markers. METHODS: The SPs from luminal-type BC were analysed for BC T-IC characteristics, including human epidermal growth factor receptor 2 (HER2), ERalpha, IGFBP7 expression and their ability to initiate tumours in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice. Pharmacological modulators were used to assess the effects of HER2 signalling and breast cancer-resistance protein (BCRP) expression on SPs. RESULTS: The SP was more prevalent in the luminal subtype of BC compared with the basal subtype. HER2 expression was significantly correlated with the occurrence of an SP (r(2)=0.75, P=0.0003). Disappearance of SP in the presence of Ko143, a specific inhibitor of the ATP-binding cassette transporter BCRP, suggests that BCRP is the predominant transporter expressed in this population. The SP also decreased in the presence of HER2 signalling inhibitors AG825 or trastuzumab, strengthening the notion that HER2 contributed to the SP phenotype, likely through downstream AKT signalling. The SP cells from luminal-type MCF-7 cells with enforced expression of HER2, and primary cells with luminal-like properties from a BC patient, displayed enrichment in cells capable of repopulating tumours in NOD/SCID mice. Engraftment of SP cells was inhibited by pretreatment with AG825 or by in vivo treatment with trastuzumab. INTERPRETATION: Our findings indicate an important role of HER2 in regulating SP and hence T-ICs in BC, which may account for the poor responsiveness of HER2-positive BCs to chemotherapy, as well as their aggressiveness.

Phosphorylation of the ErbB3 binding protein Ebp1 by p21-activated kinase 1 in breast cancer cells.

The ErbB3 binding protein (Ebp1) is a transcriptional corepressor that inhibits the activity of proliferation-associated genes and the growth of human breast cancer cell lines. Treatment of breast cancer cells with the ErbB3 ligand heregulin (HRG) results in increased phosphorylation of Ebp1 and transcriptional repression. The p21-activated serine/threonine kinase 1 (PAK1), which plays an important role in breast cancer progression and resistance to the anti-oestrogen tamoxifen, is also activated by HRG. We therefore examined the ability of PAK1 to phosphorylate and regulate the function of Ebp1. We found that PAK1 phosphorylated Ebp1 in vitro and mapped the phosphorylation site to threonine 261. Both HRG treatment and expression of a constitutively activated PAK1 in MCF-7 breast cancer cells enhanced threonine phosphorylation of Ebp1. In MCF-7 cells, ectopically expressed Ebp1 bound endogenous PAK1 and this association was enhanced by treatment with HRG. Mutation of the PAK1 phosphorylation site to glutamic acid, mimicking a phosphorylated state, completely abrogated the ability of Ebp1 to repress transcription, inhibit growth of breast cancer cell lines and contribute to tamoxifen sensitivity. These studies demonstrate for the first time that Ebp1 is a substrate of PAK1 and the importance of the PAK1 phosphorylation site for the functional activity of Ebp1 in breast cancer cells.

Spatially resolved characterization of Xe ion irradiated LiF crystals using static field gradient NMR.

Spatially resolved (19)F and (7)Li nuclear magnetic resonance (NMR) spin-lattice relaxation rates have been measured in LiF crystals irradiated with 1.44 GeV Xe ions at fluences from 10(10) to 10(12) ions cm(-2). In addition, the F-centre concentration has been measured by optical absorption spectroscopy and the concentration of paramagnetic centres by electron paramagnetic resonance (EPR). Within the ion range, the relaxation rate turns out to increase linearly with the concentration of paramagnetic centres but super-linearly with the F-centre concentration. Beyond the ion range, the relaxation rate is still significantly enhanced compared to non-irradiated LiF.

Spatially resolved characterization of heavy ion irradiated crystals using static field gradient nuclear magnetic resonance.

Static magnetic field gradient NMR has been used for one-dimensional spatial (19)F spin-lattice relaxation profile studies (resolution of the order of 10 µm) in a LiF crystal irradiated with U ions. Technical aspects of the use of large static magnetic field gradients are discussed as well as a special data acquisition mode allowing for effectively measuring spatially resolved spin-lattice relaxation rates as low as 10(-3) s(-1). In addition to the expected enhanced spin-lattice relaxation rate within the ion range, also an enhanced rate beyond the ion range has been found.

Structural insights into antibody-mediated mucosal immunity.

The mucosal regions of the body are responsible for defense against environmental pathogens. Particularly in the lumen of the gut, antibody-mediated immune responses are critical for preventing invasion by pathogens. In this chapter, we review structural studies that have illuminated various aspects of mucosal immunity. Crystal structures of IgA1-Fc and IgA-binding fragments of the polymeric immunoglobulin receptor and Fc alphaRI, combined with models of intact IgA and IgM from solution scattering studies, reveal potential mechanisms for immune exclusion and induction of inflammatory responses. Other recent structures yield insights into bacterial mechanisms for evasion of the host immune response.

Low prevalence of hearing impairment among very low birthweight infants as detected by universal neonatal hearing screening.

OBJECTIVES: To (a) study the prevalence of hearing impairment in a cohort of very low birthweight (VLBW) infants and (b) evaluate the effectiveness of transient evoked otoacoustic emissions (TEOAE) as a first stage in-hospital hearing screening tool in this population. STUDY DESIGN: The study group was a cohort of 346 VLBW infants born in 1998-2000 at The Sheba Medical Center. The prevalence of hearing impairment in the study group was compared with that of all other newborn infants participating in a universal newborn hearing screening programme during the same period. To evaluate the effectiveness of TEOAE, a control group of 1205 healthy newborns who had no known risk factors for hearing impairment was selected. The results and follow up of hearing screening for these infants were examined retrospectively. RESULTS: Only one VLBW infant (0.3%) was diagnosed with bilateral sensory-neural hearing loss. In addition, nine infants (2.7%) were diagnosed with conductive hearing loss. Bronchopulmonary dysplasia and low Apgar score were the most significant factors for predicting the occurrence of conductive hearing loss. The percentage of VLBW infants who successfully passed the in-hospital TEOAE screening was 87.2, compared with 92.2% in the full term control group. No false negative cases were detected on follow up. CONCLUSIONS: The study shows a low incidence of sensory-neural hearing loss in a cohort of VLBW infants and a relatively high incidence of conductive hearing loss. TEOAE screening was found to be an effective first stage in-hospital hearing screening tool in this population.

Specificity and heregulin regulation of Ebp1 (ErbB3 binding protein 1) mediated repression of androgen receptor signalling.

Although ErbB receptors have been implicated in the progression of prostate cancer, little is known about proteins that may mediate their interactions with the androgen receptor (AR). Ebp1, a protein cloned via its association with the ErbB3 receptor, binds the AR and inhibits androgen-regulated transactivation of wild-type AR in COS cells. As the complement of coregulators in different cells are important for AR activity, we determined the effect of Ebp1 on AR function in prostate cancer cell lines. In addition, we examined the regulation of Ebp1 function by the ErbB3/4 ligand heregulin (HRG). In this study, we demonstrate, using several natural AR-regulated promoters, that Ebp1 repressed transcriptional activation of wild-type AR in prostate cancer cell lines. Downregulation of Ebp1 expression in LNCaP cells using siRNA resulted in activation of AR in the absence of androgen. Ebp1 associated with ErbB3 in LNCaP cells in the absence of HRG, but HRG induced the dissociation of Ebp1 from ErbB3. In contrast, HRG treatment enhanced both the association of Ebp1 with AR and also the ability of Ebp1 to repress AR transactivation. These studies suggest that Ebp1 is an AR corepressor whose biological activity can be regulated by the ErbB3 ligand, HRG.

Assessment of medial olivocochlear system function in pre-term and full-term newborns using a rapid test of transient otoacoustic emissions.

This study was conducted to investigate maturation of the medial olivocochlear efferent system (MOCS) in pre- and full-term neonates using Quickscreen (Otodynamics Ltd) and to confirm previous findings on transient otoacoustic emission (TEOAE) suppression in neonates. MOCS maturation was investigated in 46 neonates born at the Chaim Sheba Medical Center, Tel Hashomer, Israel, using Quickscreen. All neonates were normal with no family history of general or auditory disease and no risk factors for hearing impairment. MOCS function appears gradually in human pre-term neonates and is considered to reach maturity shortly after term birth. The clinical value of MOCS testing in specific populations of newborns at risk for hearing and/or brainstem function can be legitimately raised as activation of the MOCS clearly alters cochlear output. The present results can be interpreted to support the testing of infants at risk of developing abnormal MOCS function using a commercially available rapid TEOAE measurement system.

[Humoral immune response in breeding sows after vaccination with a herd-specific Chlamydophila abortus vaccine]. / Humorale Immunantwort von Zuchtsauen nach Impfung mit einer stallspezifischen Chlamydophila abortus-Vakzine.

A chlamydial vaccine efficacy trial with assessment of the clinical acceptability and serum antibody responses was performed in breeding sows. A BGM cell culture derived vaccine containing 10(8)/ml formalin-inactivated purified elementary bodies (Eb.) in sterile 0.15 M saline was prepared from Chlamydophila (Ch.) abortus strain OCHL03/99 which has been isolated in the herd from a sample of vaginal discharge. Vaccination was performed as a randomised trial with parallel treatment of a vaccinated group (25 sows) and non-vaccinated control group (20 sows). Sows received two 2.0-ml doses of vaccine intramuscularly at a three week interval. Control sows were dosed with sterile 0.15 M saline, accordingly. Serological response to vaccination was measured by ELISA with a total of 204 blood serum samples (114 from the vaccine group; 90 from the control group) using crude chlamydial LPS as the antigen. Compared to the control group, vaccinated sows showed a marked primary and secondary IgG serum antibody response following the two vaccinations. Antibody levels peaked between week 7 and 14 after priming vaccination, declined incrementally until week 27 but remained significantly higher than the corresponding sham-immune control levels and the prevaccination values of the vaccine group (p < 0.05). Western blot analysis of solubilized whole Eb. of Ch. abortus, Ch. pecorum, and Chlamydia (C.) suis with pre- and postvaccination sera confirmed that vaccination induced an antibody response preferentially against a range of 13 chlamydial antigens including the 40 kDa MOMP of Ch. abortus. Clinical side effects consisting of a transient mild local inflammatory reaction at the site of injection were observed in approx. 30% of vaccinated sows. These results provide the basis for further clinical evaluation of the Ch. abortus vaccine to protect sows from chlamydia-induced reproductive disorders.

15-PGJ2, but not thiazolidinediones, inhibits cell growth, induces apoptosis, and causes downregulation of Stat3 in human oral SCCa cells.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been linked to induction of differentiation, cell growth inhibition and apoptosis in several types of human cancer. However, the possible effects of PPARgamma agonists on human oral squamous cell carcinoma have not yet been reported. In this study, treatment with 15-deoxy-Delta(12,14)-PGJ(2) (15-PGJ(2)), a natural PPARgamma ligand, induced a significant reduction of oral squamous cell carcinoma cell growth, which was mainly attributed to upregulation of apoptosis. Interestingly, rosiglitazone and ciglitazone, two members of the thiazolidinedione family of PPARgamma activators, did not exert a growth inhibitory effect. Given the critical role that the oncogene signal transducer and activator of transcription 3 (Stat3) plays in head and neck carcinogenesis, its potential regulation by PPARgamma ligands was also examined. Treatment of oral squamous cell carcinoma cells with 15-PGJ(2) induced an initial reduction and eventual elimination of both phosphorylated and unphosphorylated Stat3 protein levels. In contrast, other PPARgamma did not induce similar effects. Our results provide the first evidence of significant antineoplastic effects of 15-PGJ(2) on human oral squamous cell carcinoma cells, which may be related to downmodulation of Stat3 and are at least partly mediated through PPARgamma-independent events.

Analysis of the expression pattern of Ebp1, an ErbB-3-binding protein.

Ebp1, a member of the PA2G4 family, was isolated as an ErbB-3-binding protein in our laboratory using yeast two hybrid analysis. Although Ebp1 mRNA is ubiquitously expressed, little is known about either the expression of Ebp1 protein in vivo or its translation initiation site. Western blotting analysis of a wide range of cell lines and primary tissue indicated that in the majority of cases Ebp1 is expressed as a single protein which migrates at 48 kDa in SDS-polyacrylamide gels. We show using epitope-tagged expression constructs that the second, not the first, in-frame ATG is used for the initiation of translation of the endogenous protein, encoding a protein predicted to be 41.5 kDa. The molecular mass of endogenous Ebp1 protein derived from mouse liver and brain was determined by mass spectrometry and the data confirm that translation of endogenous Ebp1 in tissues is initiated from the second in-frame ATG.

Regulation of the ErbB3 binding protein Ebp1 by protein kinase C.

Ebp1, a 47 kDa ubiquituously expressed protein, binds the ErbB3 receptor in human serum starved breast cancer cell lines and dissociates from ErbB3 on treatment with the ErbB3 ligand, Heregulin (HRG). However, the mechanism of Ebp1-ErbB3 association/dissociation is not understood. Since Ebp1 contains six putative Protein Kinase C serine/threonine phosphorylation sites, we examined the ability of PKC to phosphorylate Ebp1 and to regulate Ebp1-ErbB3 binding. We found that Ebp1 was basally phosphorylated in AU565 breast cancer cells on serine/threonine residues and that this phosphorylation was enhanced by heregulin treatment. Both serine and threonine residues of a GST-Ebp1 fusion protein were phosphorylated by PKC in vitro. In vivo, we demonstrated that basal Ebp1 phosphorylation was dependent upon PKC. However, HRG-induced phosphorylation of Ebp1 occurred predominantly in a PKC-independent manner. The ability of Ebp1 to associate with ErbB3 in serum-starved NIH3T3 cells overexpresssing ErbB3 was abrogated by treating cells with a PKC inhibitor. These findings suggest that PKC plays a role in regulating phosphorylation and function of Ebp1 in vivo.

Ebp1, an ErbB-3 binding protein, interacts with Rb and affects Rb transcriptional regulation.

Ebp1, an ErbB-3 binding protein, inhibits the proliferation and induces the differentiation of human breast cancer cells. The mechanisms of these effects are unknown. Rb, the product of the retinoblastoma gene, is an important modulator of cell cycle progression and cellular differentiation. We report that Rb is a binding target for Ebp1. Ebp1 was localized to both the nucleus and the cytoplasm of logarithmically growing AU565 breast cancer cells and HeLa cells as determined by confocal immunofluorescent microscopy. Ebp1 was present in Rb immunoprecipitates derived from AU565 breast cancer cells. GST-Rb also bound endogenous Ebp1. Using GST-Ebp1 constructs, we determined that the 72 C-terminal amino acids of Ebp1 were sufficient to bind Rb. Dephosphorylation of Ebp1 enhanced the interaction of Ebp1 with Rb. The overexpression of Ebp1 in MCF-7 and AU565 (Rb(+)) cells inhibited the activity of the E2F1 regulated cyclin-E promoter. Ebp1 bound E2F1 indirectly via Rb in lysates of MCF-7 cells. The interaction of Ebp1 with Rb may prove to be an important mechanism of Ebp1 induced changes in cell proliferation and differentiation.

Detection of ErbB Receptor Family Tyrosyl Phosphorylation in Ovarian Carcinoma Cells.

The epidermal growth factor receptor (EGFR) (ErbB) family of receptor tyrosine kinases is frequently overexpressed in ovarian cancer (1) . This receptor group is comprised of four members, EGFR (ErbB-1), HER2 (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). These receptors share a common molecular architecture, possessing a large glycosylated extracellular ligand-binding domain, a single hydrophobic transmembrane domain, and a cytoplasmic tyrosine kinase domain (2). Amplification and over- expression of ErbB-2 have been reported in ovarian carcinoma, where high levels of ErbB-2 directly correlate with poor clinical prognosis (3,4). Amplification of EGFR has also been reported, and may have prognostic significance (5,6). Less is known about the clinical significance of ErbB-3 (7) and ErbB-4 (8), which have been more recently identified.

Ectopic expression of the ErbB-3 binding protein ebp1 inhibits growth and induces differentiation of human breast cancer cell lines.

Ebp1, an ErbB-3 binding protein, translocates from the cytoplasm to the nucleus of human breast cancer cells after treatment with the ErbB-3 ligand, heregulin. The purpose of these studies was to examine the effects of ectopic expression of ebp1 on the biological properties of human ErbB-3-expressing breast carcinoma cell lines. Ectopic expression of ebp1 in ErbB-2, ErbB-3-expressing breast carcinoma cell lines resulted in inhibition of colony formation, a decreased proliferation rate, an accumulation of cells in the G2/M phase of the cell cycle, and suppression of growth in soft agar. Ectopic expression of ebp1 led to a more differentiated phenotype in AU565 breast cancer cells, as evidenced by increased expression of lipid droplets and of the milk protein casein. Basal phosphorylation of extracellular regulated kinases (Erks) 1 and 2, kinases activated by heregulin treatment, was also observed in ebp1 transfectants. The promoter for the intercellular adhesion molecule-1 gene, a heregulin-inducible gene, was constitutively activated in ebp1 transfectants as determined by reporter construct analysis. These data demonstrate that ectopic expression of the ErbB-3 binding protein Ebp1 inhibits proliferation and induces differentiation of ErbB-2, ErbB-3-expressing human breast carcinoma cell lines.

Interaction of the PA2G4 (EBP1) protein with ErbB-3 and regulation of this binding by heregulin.

The processes by which ErbB-3, an inactive tyrosine kinase, exerts its biological effects are poorly understood. Using the yeast two-hybrid system, we have isolated an ErbB-3 binding protein (Ebp1) that interacts with the juxtamembrane domain of ErbB-3. This protein is identical to that predicted to be encoded for by the human PA2G4 gene. Ebp1 is the human homologue of a previously identified cell cycle-regulated mouse protein p38-2G4. Two transcripts of ebp1 mRNA (1.7 and 2.2 kb) were detected in several normal human organs. The interaction of Ebp1 with ErbB-3 was examined in vitro and in vivo. The first 15 amino acids of the juxtamembrane domain of ErbB-3 were essential for Ebp1 binding in vitro. Treatment of AU565 cells with the ErbB-3 ligand heregulin resulted in dissociation of Ebp1 from ErbB-3. Ebp1 translocated from the cytoplasm into the nucleus following heregulin stimulation. These findings suggest that Ebp1 may be a downstream member of an ErbB-3-regulated signal transduction pathway.

Dominance of ErbB-1 heterodimers in lung epithelial cells overexpressing ErbB-2. Both ErbB-1 and ErbB-2 contribute significantly to tumorigenicity.

This article examines differential expression and heterodimer formation of ErbB family members in tumorigenic and nontumorigenic human bronchial epithelial cells (HBECs). This cell system was developed previously as a model for lung adenocarcinoma by overexpression of c-erbB-2 in nontumorigenic, T antigen-immortalized HBECs. Earlier studies demonstrated that a tumorigenic clone from T antigen-immortalized nontumorigenic cells overexpressing ErbB-2 endogenously produced high levels of transforming growth factor (TGF)-alpha, and that reducing TGF-alpha by 93% eliminated tumorigenicity. In the present report, comparison of ErbB species between the tumorigenic cells (E6T) and their nontumorigenic derivatives (E6TA) demonstrated all four receptors in both cell types. However, in E6TA cells, ErbB-3 and -4 were present primarily in ErbB-1 heterodimers, suggesting that ErbB-1 is a preferred heterodimer partner within this cell system, expressing endogenous ErbB receptors and ligands and overexpressing ErbB-2. The ErbB-1/-2 species was present at high levels in E6T and absent in E6TA cells. Mitogen-activated protein kinase activity was elevated in E6T relative to E6TA. Elevated activity was eliminated by blocking surface expression of either ErbB-1 or ErbB-2. Endoplasmic reticulum trapping of ErbB-1 eliminated tumorigenicity, whereas ErbB-2 internalization was selected against during tumor formation. These data demonstrate the importance of TGF-alpha-mediated signaling through the ErbB-1/-2 heterodimer in development of the tumorigenic phenotype. This work further suggests that ErbB-3 and -4 species may also contribute to tumorigenic conversion and that their expression levels may be increased by signaling initiated by TGF-alpha.