Ets regulation of the erbB2 promoter.

Evaluating the chromatinized erbB2 gene in nuclei from breast cancer cells expressing varying levels of ErbB2 transcripts, we identified a nuclease-sensitive site within a 0.22 kb region of maximum enhancer activity centered over a conserved 28 bp polypurine(GGA)-polypyrimidine(TCC) mirror-repeat and an adjacent essential Ets binding site (EBS). Promoter footprinting with nuclear extracts reveals an intense Ets hypersensitivity site at the EBS whose degree of intensity correlates with the level of cellular ErbB2 expression. In vitro mapping assays show that the supercoiled erbB2 promoter forms an internal triplex structure (Hr-DNA) at the mirror-repeat element. Mutations preventing Hr-DNA formation can enhance erbB2 promoter activity in human breast cancer cells, a result consistent with previous demonstration that Ets-erbB2 promoter complexes cannot form when the mirror-repeat is engaged in triplex binding, and new results suggesting that Ets binding induces severe promoter bending that may restrict local triplex formation. In addition to previously described erbB2-regulating breast cancer Ets factors (PEA3, ESX/Elf-3), Elf-1 is now shown to be another endogenously expressed Ets candidate capable of binding to and upregulating the erbB2 promoter. Given current strategies to transcriptionally inhibit ErbB2 overexpression, including development of novel erbB2 promoter-targeted therapeutics, an EBS-targeted approach is presented using chimeric Ets proteins that strongly repress erbB2 promoter activity.

ESX: a structurally unique Ets overexpressed early during human breast tumorigenesis.

The >30 known members of the Ets multigene family of transcriptional regulators are increasingly being recognized for their involvement in early embryonic development and late tissue maturation, directing stage-specific and tissue-restricted programs of target gene expression. Identifiable primarily by their 85 amino acid ETS DNA-binding domain and dispersed across all metazoan lineages into distinct subfamilies, Ets genes also produce malignancies in humans and other vertebrates when overexpressed or rearranged into chimeras retaining the ETS domain, suggesting that their oncogenic potential is determined by the program of target genes they regulate. Searching for Ets factors that regulate expression of the HER2/neu (c-erbB2) oncogene in human breast cancer, we identified a new epithelium-restricted Ets encoding an ETS domain homologous to the Drosophila E74/human Elf-1 subfamily, an amino-terminal region (A-region or Pointed domain) homologous to the distantly related Ets-1 subfamily, and a serine-rich box homologous to the transactivating domain of the lymphocyte-restricted High Mobility Group (HMG) protein, SOX4. Recombinant protein encoded by ESX (for epithelial-restricted with serine box) exhibits Ets-like DNA binding specificity in electrophoretic mobility shift assays and, in transient transfection assays, transactivates Ets-responsive promoter elements including that found in the HER2/neu oncogene. ESX is located at chromosome 1q32 in a region known to be amplified in 50% of early breast cancers, is heregulin-inducible and overexpressed in HER2/neu activated breast cancer cells. Tissue hybridization suggests that ESX becomes overexpressed at an early stage of human breast cancer development known as ductal carcinoma in situ (DCIS).

Involvement of the Tpl-2/cot oncogene in MMTV tumorigenesis.

We report for the first time a relationship between the Tpl-2/cot oncogene and Mouse Mammary Tumor Virus (MMTV) associated transformation of mammary gland cells. A sub-genomic library generated from a primary mammary gland tumor yielded a novel MMTV integration site which disrupted the Tpl-2/cot proto-oncogene between exons 7 and 8. Comparison of a cell line derived from normal mammary gland (comma-D) and a cell line established from an MMTV induced mammary tumor (GR) demonstrated similar rearrangements within Tpl-2/cot for the GR cells but not in the comma-D cells. These rearrangements in the cell line were accompanied by an increase in the level of Tpl-2/cot specific mRNA. This data suggests that Tpl-2/cot expression may be important in epithelial cell transformation or tumor progression.

Immunopathogenesis and physical maps of fowl adenovirus serotype 9.

Restriction endonuclease maps of the genome of fowl adenovirus (FAV) serotype 9 have been constructed for the restriction endonucleases NdeI, NotI and XbaI. The total size of the FAV-9 genome was estimated to be 44.5 kb pairs, consistent with previous reports that FAV genomes are approximately 10 kb larger than human adenovirus (HAV). The pathogenicity of this virus in day-old chickens was intermediate between the pathogenicity of the non-pathogenic and the highly pathogenic FAVs.

A V beta 4-specific superantigen encoded by a new exogenous mouse mammary tumor virus.

The superantigen (SAg) expressed by mouse mammary tumor virus (MMTV) has been shown to play an essential role in the course of the viral life cycle. In the present study, we describe a V beta 4-specific SAg encoded by a new exogenous MMTV carried by the SIM mouse strain. This is the first report of a viral or bacterial SAg reacting with mouse V beta 4+ T cells. Injection of MMTV(SIM) into adult BALB/c mice leads to a rapid and strong stimulation of V beta 4+ CD4+ T cells, followed by a slow deletion of these cells. Neonatal exposure to the virus also leads to a progressive deletion of V beta 4+ T cells. In contrast to other strong MMTV SAg, this new SAg requires the presence of major histocompatibility complex class II I-E molecules to be presented efficiently to T cells. Sequence analysis revealed a new predicted amino acid sequence in the C-terminal polymorphic region of this SAg. Furthermore, sequence comparisons to the most closely related SAg with different V beta specificities hint at the specific residues involved in the interaction with the T cell receptor.

Immunological and molecular comparison of fowl adenovirus serotypes 4 and 10.

Some discussion has centered on whether fowl adenovirus (FAV) serotypes 4 and 10 are distinct serotypes or in fact should be reclassified as a single serotype. We have undertaken a detailed characterisation of representatives of both serotypes in order to determine if types 4 and 10 should be grouped together or retained as distinct serotypes. Examination at the genomic level has revealed considerable similarities and few differences between these 2 serotypes. DNA cross-hybridization failed to distinguish between them and restriction enzyme analysis demonstrated limited sequence differences. In vivo studies demonstrated the cross-protection afforded by a natural route vaccination with serotype 4 FAV when chickens were challenged with serotype 10 FAV. On the basis of these studies it is suggested that these FAV serotypes be combined in future FAV classification. Physical maps for both serotype 4 and 10 have been constructed using the restriction enzymes Hpa I, Dra I, Nde I, Xba I and Not I for both serotypes and in addition Eco RI, Sfi I, Sma I and BglII for serotype 10.

Replication cycle and associated biosynthetic reactions for a non-oncogenic avian adenovirus.

The replication cycle of the non-oncogenic fowl adenovirus serotype 10 (FAV-10) has been examined. The onset of viral DNA synthesis was shown to commence at about 10 h postinfection (hpi) defining the early period of viral replication as prior to this time and the late phase as that time following the initiation of DNA replication. Virus titre rapidly increased between 18 and 24 hpi with maximum virus yield between 28 and 30 hpi. The late phase transcription profiles of the FAV-10 genome from 10 hpi to 24 hpi were determined. Late translation of virus protein began about 14 hpi increasing rapidly between 18 and 30 hpi.

Serological relationships within the group E fowl adenoviruses.

Antisera were raised in chickens to six group E fowl adenoviruses (FAV) which have been divided into a highly virulent (hypervirulent) and a mildly virulent subgroup using restriction endonuclease analysis. Virus neutralisations showed that these two distinct restriction endonuclease groups were distinguishable serologically, and indicated a possible vaccine candidate for use against the hypervirulent FAV. The suitability of this candidate was established in challenge experiments where vaccination with this virus protected against challenge from another hypervirulent virus as well as one of the mildly virulent FAV.

Molecular characterization of highly virulent fowl adenoviruses associated with outbreaks of inclusion body hepatitis.

Restriction enzyme analysis of DNA was used to characterize fowl adenoviruses (FAVs) consistently associated with outbreaks of acute inclusion body hepatitis. When low doses of these FAVs were administered via a natural route to chickens they caused IBH. A strong genomic relationship was demonstrated between these virulent FAVs. In contrast, the genomes of serologically related, but non-virulent or mildly virulent FAVs were found to differ substantially from those of the virulent FAVs.

Virus-neutralizing and passively protective monoclonal antibodies to infectious bursal disease virus of chickens.

Murine monoclonal antibodies (MAbs) were produced to assist in the identification and characterization of the virus-neutralizing epitopes of infectious bursal disease virus (IBDV). Only MAbs that reacted in Western blotting with viral protein 2 (VP2) or immunoprecipitated VP2 neutralized the infectivity of the virus in cell culture and passively protected young chickens from infection. Three of the neutralizing MAbs did not react with denatured viral proteins. Additivity enzyme-linked immunosorbent assays indicated that the six virus-neutralizing MAbs recognized two spatially independent epitopes. The ability of two of the virus-neutralizing MAbs to neutralize a variant of IBDV that had escaped neutralization by all the other MAbs confirmed the existence of two distinct neutralizing epitopes. The results support the hypothesis that there are at least two non-overlapping epitopes recognized by the virus-neutralizing MAbs reported in this study, although these may still be within one conformational site on VP2 of IBDV.

A conformational immunogen on VP-2 of infectious bursal disease virus that induces virus-neutralizing antibodies that passively protect chickens.

VP-2b, a major structural protein of infectious bursal disease virus (IBDV), and its precursor protein VP-2a were separated in a soluble form from the supernatant of ultracentrifuged viruses by using a monoclonal antibody specific for VP-3, the other major structural protein, to remove soluble VP-3 and remaining virus particles. The native VP-2a/2b inhibited the majority of virus-neutralizing (VN) activity in chicken anti-IBDV sera and chickens immunized with VP-2a/2b produced VN antibodies that passively protected susceptible chickens from infection. However, the separated VP2a/2b was not as immunogenic as intact virus particles. VP-2a/2b would appear to contain a conformational epitope which is destroyed by SDS and boiling and which may prove to be of critical importance in a subunit vaccine against type 1 IBDV.

Expression in Escherichia coli of cDNA fragments encoding the gene for the host-protective antigen of infectious bursal disease virus.

The larger segment of the IBDV genome codes for a 32-kDa host-protective antigen. Inserts from a cDNA library in pBR 322, containing overlapping cDNA fragments of varying sizes and covering the entire large segment of the IBDV genome, were subcloned into a mixture of expression vectors pUR 290, 291, and 292. Clones expressing the host-protective antigen, or parts of it, were identified by an immunoblot assay and the fusion proteins were further characterized by Western blot analysis using a monoclonal antibody specific for the 32-kDa polypeptide. Hybridization of inserts from expressing clones to the original cDNA library led to the identification of the region of the IBDV genome that codes for the 32-kDa host-protective antigen. Clone D1 which encodes approximately 50% and clone D6 which encodes the entire 32-kDa protein were selected for further studies. The fusion proteins from clones D1 and D6 were affinity purified and tested for their immunogenicity in chickens. Both fusion proteins induced the synthesis of antibodies in both primed and unprimed chickens that reacted specifically with denatured 32-kDa viral protein, but less well with intact virus. It was concluded that the response to the fusion proteins was to linear rather than conformational epitopes on the 32-kDa viral protein.