WW domain containing E3 ubiquitin protein ligase 1 targets the full-length ErbB4 for ubiquitin-mediated degradation in breast cancer.

ErbB4, a member of the epidermal growth factor receptor family, plays a role in normal breast and breast cancer development by regulating mammary epithelial cell proliferation, survival and differentiation. In this study, we show that WWP1, a C2-WW-HECT type E3 ubiquitin ligase, binds, ubiquitinates and destructs ErbB4-CYT1, but much less efficiently for CYT2, isoforms (both JMa and JMb). The protein-protein interaction occurs primarily between the first and third WW domains of WWP1 and the second PY motif of ErbB4. Knockdown of WWP1 by two different small interfering RNAs increases the endogenous ErbB4 protein levels in both MCF7 and T47D breast cancer cell lines. In addition, overexpression of the wild type, but not the catalytic inactive WWP1, dramatically decreases the endogenous ErbB4 protein levels in MCF7. Importantly, we found that WWP1 negatively regulates the heregulin-beta1-stimulated ErbB4 activity as measured by the serum response element report assay and the BRCA1 mRNA expression. After a systematic screening of all WWP1 family members by small interfering RNA, we found that AIP4/Itch and HECW1/NEDL1 also negatively regulate the ErbB4 protein expression in T47D. Interestingly, the protein expression levels of both WWP1 and ErbB4 are higher in estrogen receptor-alpha-positive than in estrogen receptor-alpha-negative breast cancer cell lines. These data suggest that WWP1 and its family members suppress the ErbB4 expression and function in breast cancer.

Alternative splicing of the ErbB-4 cytoplasmic domain and its regulation by hedgehog signaling identify distinct medulloblastoma subsets.

Medulloblastoma (MB) results from aberrant development of cerebellar neurons in which altered hedgehog (Hh) signalling plays a major role. We investigated the possible influence of Hh signalling on ErbB-receptor expression in MB, in particular that of the ErbB-4 CYT-1 and CYT-2 isoforms generated by alternative splicing of the cytoplasmic domain. ErbB-4 expression was downregulated in Hh-induced MBs from Patched-1(+/-) mice. Hh signalling (reflected by enhanced expression of the Gli1 transcription factor) inhibited ErbB-4 expression in mouse cerebellar granule progenitors and human MB cells. Analysis of 26 human primary MBs revealed a subset of 11 tumors characterized by low Gli1 levels, upregulated ErbB-4 expression and increased CYT-1:CYT-2 ratios. Interestingly, CYT-1 and Gli1 levels were inversely correlated. ErbB-4 CYT-1 and CYT-2 had different phenotypic effects in cultured MB cells: in response to neuregulin treatment, CYT-2 overexpression inhibited proliferation whereas CYT-1, which includes a phosphatidylinositol 3-kinase (PI3K)-binding site that is missing in CYT-2, enhanced resistance to starvation- and etoposide-induced apoptosis by activating PI3K/Akt signalling. CYT-1:CYT-2 ratios displayed correlation with tumor histotype and ErbB-2 levels, which are established prognostic indices for MB. These findings demonstrate that low-level Hh signalling in human MB is associated with the selective maintenance of high ErbB-4 CYT-1 expression, an alteration that exerts tumor-promoting effects.

Negative regulation of receptor tyrosine kinase signals.

In Metazoans a number of cellular functions are controlled by receptor tyrosine kinases (RTKs) during development and in postnatal life. The execution of these programs requires that signals of adequate strength are delivered for the appropriate time within precise spatial boundaries. Several RTK inhibitors have been identified in invertebrate and mammalian organisms. Because they are involved in tuning and termination of receptor signals, negative regulators of RTK activity fulfill a fundamental function in the control of receptor signaling.

NRG-3 in human breast cancers: activation of multiple erbB family proteins.

Ligands of the EGF/Heregulin family control the growth of epithelial cells by binding to receptors of the erbB family. By searching a large database of cDNA sequences at Human Genome Sciences Inc. we have identified a new encoded protein sequence containing all the conserved elements of the EGF/Heregulin family. The same sequence has recently been independently identified as NRG-3. The EGF-like domain of NRG-3 was generated as a recombinant protein in E. coli and used to test the specificity of receptor binding. In human breast cancer cells and in 32D cells transfected by erbB family members, NRG-3 activated multiple erbB family members. These include EGF receptor (erbB1) and erbB4 when expressed individually and erbB2 and erbB3 when expressed together. Recombinant NRG-3 altered the growth of human breast cancer cells growing in vitro. NRG-3 was expressed in cell lines derived from breast cancer. These results indicate that NRG-3 is a potential regulator of normal and malignant breast epithelial cells in vivo.

ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network.

The recently isolated second family of neuregulins, NRG2, shares its primary receptors, ErbB-3 and ErbB-4, and induction of mammary cell differentiation with NRG1 isoforms, suggesting functional redundancy of the two growth factor families. To address this possibility, we analyzed receptor specificity of NRGs by using an engineered cellular system. The activity of isoform-specific but partly overlapping patterns of specificities that collectively activate all eight ligand-stimulatable ErbB dimers was revealed. Specifically, NRG2-alpha [corrected], like NRG1-beta [corrected], emerges as a narrow-specificity ligand, whereas NRG2-beta [corrected] is a pan-ErbB ligand that binds with different affinities to all receptor combinations, including those containing ErbB-1, but excluding homodimers of ErbB-2. The latter protein, however, displayed cooperativity with the direct NRG receptors. Apparently, signaling by all NRGs is funneled through the mitogen-activated protein kinase (MAPK). However, the duration and potency of MAPK activation depend on the identity of the stimulatory ligand-receptor ternary complex. We conclude that the NRG-ErbB network represents a complex and nonredundant machinery developed for fine-tuning of signal transduction.

ErbB-4 ribozymes abolish neuregulin-induced mitogenesis.

The epidermal growth factor-like receptor tyrosine kinase (ErbB) family is frequently overexpressed in a variety of human carcinomas, including breast cancer. To assist in characterizing the role of ErbB-4 in breast cancer, we generated three specific hammerhead ribozymes targeted to the ErbB-4 mRNA. These ribozymes, Rz6, Rz21, and Rz29, efficiently catalyzed the specific cleavage of ErbB-4 message in a cell-free system. We demonstrated that the neuregulin-induced mitogenic effect was abolished in ribozyme Rz29- and Rz6-transfected 32D/ErbB-4 cells. Inhibition of mitogenesis was characterized by ribozyme-mediated down-regulation of ErbB-4 expression. In addition, we provide the first evidence that different threshold levels of ErbB-4 expression and activation correlate with different responses to neuregulin stimulation. High levels of ErbB-4 expression, phosphorylation, and homodimerization are necessary for neuregulin-stimulated, interleukin 3-independent cell proliferation in the 32D/E4 cells. In the case of Rz29-transfected 32D/E4 cells, low levels of ErbB-4 expression allowed neuregulin-induced phosphorylation but were insufficient to couple the activated receptor to cellular signaling. Furthermore, expression of the functional ErbB-4 ribozyme in T47D human breast carcinoma cells led to a down-regulation of endogenous ErbB-4 expression and a reduction of anchorage-independent colony formation. These studies support the use of ErbB-4 ribozymes to define the role of ErbB-4 receptors in human cancers.

ErbB2 expression increases the spectrum and potency of ligand-mediated signal transduction through ErbB4.

Interleukin 3-dependent murine 32D cells do not detectably express members of the ErbB receptor family and do not proliferate in response to known ligands for these receptors. 32D transfectants were generated expressing human ErbB4 alone (32D.E4) or with ErbB2 (32D.E2/E4). Epidermal growth factor (EGF), neuregulin 1-beta (NRG1-beta), betacellulin (BTC), transforming growth factor-alpha (TGF-alpha), heparin binding-EGF (HB-EGF), and amphiregulin were analyzed for their ability to mediate mitogenesis in these transfectants. 32D.E4 responded mitogenically to NRG1-beta and BTC. Surprisingly, EGF also induced significant DNA synthesis and TGF-alpha was negligibly mitogenic on 32D.E4 cells, whereas HB-EGF and amphiregulin were inactive. Although coexpression of ErbB2 with ErbB4 in 32D.E2/E4 cells did not significantly alter DNA synthesis in response to NRG1-beta or BTC, it greatly enhanced mitogenesis elicited by EGF and TGF-alpha and unmasked the ability of HB-EGF to induce proliferation. EGF-related ligands that exhibited potent mitogenic activity on 32D.E2/E4 cells at low concentrations induced adherence, morphological alterations, and up-regulation of the Mac-1 integrin and FcgammaRII/III at higher concentrations. While 125I-EGF could be specifically crosslinked to both 32D.E4 and 32D.E2/E4 cells, its crosslinking capacity was greatly enhanced in the cotransfected cells. The ability of the various ligands to mediate proliferation and/or adhesion in the two transfectants correlated with their capacity to induce substrate tyrosine phosphorylation and to initiate and sustain activation of mitogen-activated protein kinase. We conclude that the ability of ErbB4 to mediate signal transduction through EGF-like ligands is broader than previously assumed and can be profoundly altered by the concomitant expression of ErbB2.

Epiregulin is a potent pan-ErbB ligand that preferentially activates heterodimeric receptor complexes.

The ErbB signaling network consists of four transmembrane receptor tyrosine kinases and more than a dozen ligands sharing an epidermal growth factor (EGF) motif. The multiplicity of ErbB-specific ligands is incompletely understood in terms of signal specificity because all ErbB molecules signal through partially overlapping pathways. Here we addressed the action of epiregulin, a recently isolated ligand of ErbB-1. By employing a set of factor-dependent cell lines engineered to express individual ErbBs or their combinations, we found that epiregulin is the broadest specificity EGF-like ligand so far characterized: not only does it stimulate homodimers of both ErbB-1 and ErbB-4, it also activates all possible heterodimeric ErbB complexes. Consistent with its relaxed selectivity, epiregulin binds the various receptor combinations with an affinity that is approximately 100-fold lower than the affinity of ligands with more stringent selectivity, including EGF. Nevertheless, epiregulin's action upon most receptor combinations transmits a more potent mitogenic signal than does EGF. This remarkable discrepancy between binding affinity and bioactivity is permitted by a mechanism that prevents receptor down-regulation, and results in a weak, but prolonged, state of receptor activation.

Recombinant heregulin-Pseudomonas exotoxin fusion proteins: interactions with the heregulin receptors and antitumor activity in vivo.

Growth factor receptors provide unique opportunities for development of targeted anticancer therapy. Members of the type I receptor tyrosine kinase family, including epidermal growth factor (EGF) receptor (EGFR) and ErbB-2/neu, are often overexpressed in various human cancer cells, including breast. Recently, it has been shown that both ErbB-3 and ErbB-4 are receptors for heregulin (HRG)/Neu differentiation factor. Eight chimeric toxins composed of the extracellular and EGF-like domains of four different HRG isoforms and truncated Pseudomonas exotoxin (PE38KDEL) were constructed. The fusion proteins exhibited activity similar to the native HRG in inducing ErbB receptors phosphorylation. The EGF-like domain of HRG13 and HRGbeta2 fused to PE38KDEL showed the highest cytotoxic activity, with a IC50 of < or = 0.001 ng/ml. The alpha isoforms that were fused to PE38KDEL were 100-fold less active than the beta isoforms. The HRG-Pseudomonas exotoxin (PE) toxins show extremely high activity against cells expressing ErbB-4 receptor, alone or together with other members of the ErbB receptor family. Cells that do not express ErbB-4 but express ErbB-3 receptor, together with the ErbB-2 or EGFR, exhibited moderate sensitivity to HRG-PE toxins. HRG-PE toxins have little or no activity against cells expressing EGFR, ErbB-2, or ErbB-3 alone. More than an 80% tumor regression was achieved by intratumor injection of 1 microg of fusion proteins per day for 5 days. Continuous i.p. administration of EGF-like domain of HRGbeta1-PE38KDEL for 7 days via a miniosmotic pump at a dose of 40 microg/kg/day inhibited the growth of ErbB-4 receptor positive but not ErbB-4 receptor negative cell lines in athymic nude mice. We conclude that there is therapeutic potential of HRG-PE toxins in the therapy of cancers overexpressing the ErbB-4 or ErbB-2 plus ErbB-3 receptors.

Epidermal growth factor and betacellulin mediate signal transduction through co-expressed ErbB2 and ErbB3 receptors.

Interleukin-3 (IL-3)-dependent murine 32D cells do not detectably express epidermal growth factor receptors (EGFRs) and do not proliferate in response to EGF, heregulin (HRG) or other known EGF-like ligands. Here, we report that EGF specifically binds to and can be crosslinked to 32D transfectants co-expressing ErbB2 and ErbB3 (32D.E2/E3), but not to transfectants expressing either ErbB2 or ErbB3 individually. [125I]EGF-crosslinked species detected in 32D. E2/E3 cells were displaced by HRG and betacellulin (BTC) but not by other EGF-like ligands that were analyzed. EGF, BTC and HRG also induced receptor tyrosine phosphorylation, activation of downstream signaling molecules and proliferation of 32D.E2/E3 cells. 32D transfectants were also generated which expressed an ErbB3-EGFR chimera alone (32D.E3-E1) or in combination with ErbB2 (32D. E2/E3-E1). While HRG stimulation of 32D.E3-E1 cells resulted in DNA synthesis and receptor phosphorylation, EGF and BTC were inactive. However, EGF and BTC were as effective as HRG in mediating signaling when ErbB2 was co-expressed with the chimera in the 32D.E2/E3-E1 transfectant. These results provide evidence that ErbB2/ErbB3 binding sites for EGF and BTC are formed by a previously undescribed mechanism that requires co-expression of two distinct receptors. Additional data utilizing MDA MB134 human breast carcinoma cells, which naturally express ErbB2 and ErbB3 in the absence of EGFRs, supported the results obtained employing 32D cells and suggest that EGF and BTC may contribute to the progression of carcinomas that co-express ErbB2 and ErbB3.

PLC-gamma activation is required for PDGF-betaR-mediated mitogenesis and monocytic differentiation of myeloid progenitor cells.

To investigate the molecular mechanisms mediating hematopoietic cell differentiation and mitogenesis by activation of the platelet-derived growth factor beta receptor (PDGF-betaR), the wild type PDGF-betaR (PDGF-betaRWT) and tyrosine to phenylalanine mutants of the PDGF-betaR, including F751, F966, F970, F1009, F1021 and F1009/F1021 were overexpressed in FDC-P2 myeloid progenitor cells by retroviral-mediated gene transfer. Stimulation of PDGF-betaRWT and F966, F970 and F1009 infectants with PDGF-BB led to the increased expression of monocytic differentiation markers. In contrast, activation of PDGF-betaR in the parental line or the F1021 or F1009/F1021 mutant infectants failed to induce monocytic differentiation. PDGF-BB stimulation of PDGF-betaRWT, F751, F966, F970 and F1009 infectants led to pronounced DNA synthesis, whereas F1021 and F1009/F1021 infectants did not reveal any increase in mitogenesis when compared to that of the FDC-P2 line. While PDGF stimulation of FDC-P2 cells overexpressing PDGF-betaRWT led to a pronounced increase in inositol phosphate formation due to phospholipase C-gamma (PLC-gamma) activation, PDGF-BB induced phosphoinositol hydrolysis was completely abolished in the F1021 and F1009/F1021 infectants. GF 109203X, a specific inhibitor of protein kinase C (PKC) activation, fully blocked PDGF-betaR-mediated monocytic differentiation and mitogenesis. Taken together, these results suggest that stimulation of the PDGF-betaR signaling pathway can mediate monocytic differentiation when PDGF-betaR is expressed at sufficient levels and that activation of PLC-gamma and PKC plays a pivotal role in PDGF-betaR-mediated differentiation and mitogenesis in FDC-P2 cell system.

Identification of tyrosine 187 as a protein kinase C-delta phosphorylation site.

Protein kinase C-delta (PKC-delta) has been demonstrated to be phosphorylated on tyrosine residue(s) in many different biological systems (Li, W., Yu, J.-C., Michieli, P., Beeler, J. F., Ellmore, N., Heidaran, M. A., and Pierce, J. H. (1994) Mol. Cell. Biol. 14, 6727-6735; Li, W., Mischak, H., Yu, J.-C., Wang, L.-M., Mushinski, J. F., Heidaran, M. A., and Pierce, J. H. (1994) J. Biol. Chem. 269, 2349-2352; Denning, M. F., Dlugosz, A. A., Howett, M. A., and Yuspa, S. H. (1993) J. Biol. Chem. 268, 26079-26081). Tyrosine phosphorylation of PKC-delta has also been shown to occur in vitro when purified PKC-delta is coincubated with different tyrosine kinase sources. However, the tyrosine phosphorylation site(s) is currently unknown and the exact effect of this phosphorylation on its serine/threonine kinase activity and biological functions is still controversial. To directly investigate the potential role of PKC-delta tyrosine phosphorylation, tyrosine 187 was converted to phenylalanine (PKC-deltaY187F) by site-directed mutagenesis, and expression vectors containing PKC-deltaY187F cDNAs were transfected into both 32D myeloid progenitor cells and NIH 3T3 fibroblasts. The results showed that tyrosine 187 of PKC-delta became phosphorylated in vivo in response to 12-O-tetradecanoylphorbol-13-acetate stimulation or platelet-derived growth factor receptor activation. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that one phosphopeptide was absent in PKC-deltaY187F when compared to wild type PKC-delta, further substantiating that tyrosine 187 of PKC-delta is phosphorylated in vivo. Although the phosphotyrosine content of PKC-deltaY187F was reduced compared with PKC-deltaWT, the kinase activity of PKC-deltaY187F toward a PKC-delta substrate was not altered. Moreover, 12-O-tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells was not affected by expression of the PKC-deltaY187F mutant. Taken together, these results suggest that tyrosine phosphorylation of PKC-delta on 187 may not influence PKC-delta activation and known functions.

Expression of an ATP binding mutant of PKC-delta inhibits Sis-induced transformation of NIH3T3 cells.

In an effort to determine the role of protein kinase C-delta (PKC-delta) in cellular transformation mediated by the sis proto-oncogene, we cotransfected expression vectors containing cDNAs that encode for c-sis with an ATP binding mutant of PKC-delta (PKC-delta K376R) or wild type PKC-delta (PKC-delta WT) into NIH3T3 cells. Our results showed that expression of PKC-delta K376R severely impaired Sis-induced focus formation, whereas cotransfection of PKC-delta WT cDNA had no effect on Sis-mediated transformation. Consistent with this result, PKC-delta K376R expression also inhibited PDGF-BB-mediated anchorage-independent colony formation. While cotransfection of a vector containing a dominant negative mutant of ras (N17 ras) cDNA potently inhibited Sis-induced transformation, the expression of PKC-delta K376R did not block transformation mediated by v-H-Ras or v-Raf. In addition, PDGF-BB-induced Raf and mitogen-activated protein kinase activation, which are known to be downstream molecules in the Ras cascade, were not affected by the expression of PKC-delta K376R, indicating that PKC-delta and Ras are segregated in mediating Sis-induced transformation. Interestingly, expression of PKC-delta K376R strongly reduced TPA responsive element (TRE) transactivation induced by PDGF stimulation, suggesting that activation of TRE-containing genes, which may be involved in Sis-mediated transformation, are negatively regulated by expression of PKC-delta K376R.

All ErbB receptors other than the epidermal growth factor receptor are endocytosis impaired.

Four transmembrane tyrosine kinases constitute the ErbB receptor family: the epidermal growth factor (EGF) receptor, ErbB-2, ErbB-3, and ErbB-4. We have measured the endocytic capacities of all four members of the EGF receptor family, including ErbB-3 and ErbB-4, which have not been described previously. EGF-responsive chimeric receptors containing the EGF receptor extracellular domain and different ErbB cytoplasmic domains (EGFR/ErbB) have been employed. The capacity of these growth factor-receptor complexes to mediate 125I-EGF internalization, receptor down-regulation, receptor degradation, and receptor co-immunoprecipitation with AP-2 was assayed. In contrast to the EGF receptor, all EGFR/ErbB receptors show impaired ligand-induced rapid internalization, down-regulation, degradation, and AP-2 association. Also, we have analyzed the heregulin-responsive wild-type ErbB-4 receptor, which does not mediate the rapid internalization of 125I-heregulin, demonstrates no heregulin-regulated receptor degradation, and fails to form association complexes with AP-2. Despite the substantial differences in ligand-induced receptor trafficking between the EGF and ErbB-4 receptors, EGF and heregulin have equivalent capacities to stimulate DNA synthesis in quiescent cells. These results show that the ligand-dependent down-regulation mechanism of the EGF receptor, surprisingly, is not a property of any other known ErbB receptor family member. Since endocytosis is thought to be an attenuation mechanism for growth factor-receptor complexes, these data imply that substantial differences in attenuation mechanisms exist within one family of structurally related receptors.

Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas.

In the present study we demonstrate that erbB-3 and erbB-2 cooperate in neoplastic transformation. Under conditions in which neither gene alone induced transformation, they readily transformed NIH3T3 cells if co-expressed. Furthermore, at high expression levels of ErbB2 which cause transformation, ErbB3 enhanced focus formation by one order of magnitude. Synergy required an intact ErbB2 extracellular domain and tyrosine kinase activity. Cooperation between ErbB3 and ErbB2 involved heterodimerization and increased tyrosine phosphorylation of ErbB3. Signaling by the heterodimer resulted in increased PI 3-kinase recruitment as well as quantitative and qualitative differences in substrate phosphorylation. Evidence for signaling by an active ErbB3-ErbB2 heterodimer in four mammary tumor cell lines indicated relevance of this mechanism for human neoplasia. Our detection of the NDF/heregulin transcript in NIH3T3 cells implicates an autocrine loop involving this ligand in signaling by the ErbB3-ErbB2 heterodimer in the model system, whereas heregulin-independent mechanisms likely exist for cooperative signaling by ErbB3 and ErbB2 chronically activated in some human mammary carcinomas.

Localization of the human HER4/erbB-4 gene to chromosome 2.

The HER4/erbB-4 gene has been isolated as the fourth member of the human EGFR subfamily of tyrosine kinases and has been reported to encode a receptor for NDF/heregulin. In the present study we determined the chromosomal location of the HER4/erbB-4 gene within the human genome. Using human cDNA probes in fluorescence in situ hybridization (FISH), we mapped the HER4/erbB-4 gene to human chromosome 2q33.3-34. This finding established that also the HER4/erbB-4 gene is located in close vicinity of homeobox and collagen gene loci, as is the case for the related EGFR, erbB-2/neu and erbB-3. Aberrations of this chromosomal region associated with T cell leukemias and lymphomas as well as alveolar rhabdomyosarcomas raise the possibility that HER4/erbB-4 might be activated in these tumour types.

High cell kinetics is associated with amplification of the int-2, bcl-1, myc and erbB-2 proto-oncogenes and loss of heterozygosity at the DF3 locus in primary breast cancers.

Cell kinetics is a predictive parameter of breast-cancer aggressiveness, and mutations occurring in mammary tumorigenesis may favor uncontrolled cell proliferation. In this study, cell kinetics, clinico-pathological characteristics and genetic alterations at the int-2, bcl-1, c-myc, c-erbB-2, and DF3 loci were analyzed and correlated in 54 primary breast carcinomas. The occurrence of mutations at more than one locus was also studied. Tumor-proliferative activity was evaluated by determination of the thymidine labeling index (TLI). Amplification (AMP) of int-2 was observed in 11.2%, of bcl-1 in 9.4%, of c-myc in 5.7% and of c-erbB-2 in 8.6% of the carcinomas. Loss of heterozygosity (LOH) at the DF3 locus was detected in 13.9% of the tumors. Genetic alterations demonstrated a significant association with patient's age and high TLI values. AMP and LOH+AMP did not appear to be statistically related to histotype, histological grade, tumor size or lymph-node status. Alone, allele loss at the DF-3 locus was not significantly associated with any of the clinico-pathological characteristics studied. Alterations at more than one locus, including int-2/bcl-1, int-2/c-myc, int-2/bcl-1/c-erbB-2, and c-myc/DF3, were detected in 11.1% of the tumors. Multiple mutations were found only in less differentiated tumors, which included the 2 cases from the youngest patients of the series.

p53 expression and genetic evidence for viral infection in intraepithelial neoplasia of the uterine cervix.

Infections with high-risk strains of human papillomaviruses (HPVs) and with herpes simplex virus type 2 (HSV 2), as well as inactivation of the p53 tumor suppressor gene, are important cofactors in cervical carcinogenesis. We analyzed 41 paraffin-embedded cervical intraepithelial lesions, including 25 cases of low-grade cervical intraepithelia neoplasia (CIN), and 16 cases of high-grade CIN for the presence of HPV 16/18 and HSV 2 genomic sequences and for the nuclear accumulation of the p53 protein. HPV 16 DNA was detected in 24.% of low-grade CINs and in 43.7% of high-grade CINs. HPV 18 was found only in 8.% of low-grade CINs. None of the cases tested scored positive for HSV 2 DNA. Nuclear accumulation of p53 was found in 4% of low-grade CINs, and in 31.2% of high-grade CINs, including 57.1% of the lesions that were positive for HPV 16. These results indicate that HPV 16 infection was over sixfold more common than HPV 18 infection and that p53 overexpression was significantly associated with high-grade lesions.

Expression of epitopes of the tumour-associated glycoprotein 72 and clinicopathological correlations in mammary carcinomas.

We analysed the immunohistochemical expression pattern of the distinct carbohydrate epitopes of the TAG-72 molecule, defined by the monoclonal antibodies (MAbs) B72.3, CC-49 and CC-83, in 92 breast carcinomas of different histological type, and in other histological components identified in the mammary tissue samples studied. The results were correlated with the clinico-pathological characteristics of the tumours, and with their proliferative activity, assessed by thymidine labelling index (TLI). Expression of the TAG-72 epitopes was detected in all the tumour histotypes analysed, but patterns of immunoreactivity tended to vary in relation to type and level of differentiation. The comparative analysis of the reactivities of the three anti-TAG-72 MAbs revealed differences in their ability to recognise neoplastic lesions. MAb CC-49 reacted with the highest percentage of tumours (82%), and also tended to yield the highest percentages of immunoreactive cancer cells, while B72.3 and CC-83 reacted with lower percentages of tumours (respectively, 55 and 51%), and identified lower percentages of immunoreactive cells. High levels of expression of the three TAG-72 epitopes were detected in areas of in situ ductal carcinoma. Comparatively lower levels of immunohistochemical positivity were found in atypical epithelial hyperplasia, normal mammary epithelium and epithelium with cystic disease. TAG-72 epitope expression was correlated with prognostic parameters. The synchronous expression of the three epitopes significantly correlated with large tumour size (> 2 cm), and with high histological grade. No correlations could be demonstrated between TAG-72 phenotypes and nuclear grade, lymph node status and proliferative activity (high versus low).

Detection of antibodies to human immunodeficiency virus type I by using synthetic peptides and time-resolved fluoroimmunoassay (TR-FIA).

A simple, rapid, reproducible and sensitive peptide-Time-Resolved-Fluoroimmunoassay (TR-FIA) method is described which allows the detection of antibodies to the Human Immunodeficiency Virus type 1 (HIV-1). By using a panel of synthetic peptide antigens that covered env, gag and pol amino acid sequences, a 20 amino acid peptide (GIWGCSGKLICTTAVPWNAS) describing an immunodominant and conserved domain on the gp41 region of the BH10 clone was found to be the most reactive in this study. Optimal conditions for antigen concentration, serum dilution and incubation time were established. The peptide-TR-FIA is specific, as assessed by testing HIV-1 positive sera which included samples from AIDS, ARC patients and HIV-positive drug users. The test was used to detect HIV antibodies in 250 well characterized HIV-1 positive sera and 50 normal sera. Peptide-TR-FIA results indicate that the env peptide was highly reactive with HIV-positive sera showing a sensitivity of 100%. None of the 50 control sera showed positive reactivity against the synthetic peptide. Furthermore the peptide-TR-FIA allowed a fine titration of antibodies to defined epitopes of immunodominant HIV structural proteins that usually cannot be achieved by peptide-ELISA assays.