Andre T. Baron

EGF/ErbB Receptor Family in Ovarian Cancer

In summary, the EGF/ErbB family of receptor tyrosine kinases has been shown to play a key role in normal ovarian follicle development, and cell growth regulation of the ovarian surface epithelium. Disregulation of these normal growth regulatory pathways, including overexpression and/or mutation of EGFR/ErbB receptor family members, as well as elements of their downstream signalling pathways, have been shown to contribute to the etiology and progression of epithelial ovarian cancer. It is, therefore, not surprising that these gene products, and their related soluble receptor isoforms may have clinical utility as tumor and/or serum biomarkers of disease activity. Moreover, since several of these soluble receptor isoforms have potent growth inhibitory activity, and are naturally occurring in the circulation, they are ideal candidates for the development of novel therapeutics for the treatment of ovarian cancer patients.

Centrin is a component of the periocentriola lattice

Summary— Here, we use three polyclonal anticentrin antisera designated 08/28, 26/14‐1, and 26/14‐2 to further characterize the pericentriolar lattice of metazoan cells. all of these antibodies give an indistinguishable localization pattern that consists of a constellation of pericentrosomal spots. In QT6 cells these spots are few in number and closely associated with the centriolar region, whereas in PtK2 cells they are more numerous and distributed further from the point of microtubule focus. In mitotic cells, centrin is localized to the spindle poles and spindle apparatus. We demonstrate here that the pericentriolar lattice of PtK2 and QT6 cells is, in part, composed of proteins characterized by acidic pIs (4.4 to 5.4), low molecular mass (Mr 18 500–21000), and calcium‐binding; these attributes and the immunoreactivity of these proteins to anticentrin antibodies indicate that they are centrin isoforms of metazoan cells. Finally, we confirm our earlier observation that PtK2 cells contain a centrin‐related protein of Mr 165000; QT6 cells also contain centrin‐related proteins (Mr 64000–165000). We conclude that centrin is a component of the pericentriolar lattice of higher eukaryotic centrosomes.

A sandwich type Acridinium-Linked ImmunoSorbent Assay (ALISA) detects soluble ErbB1 (sErbB1) in normal human sera

The epidermal growth factor receptor (ErbB1) is overexpressed in various human tumor-derived cell lines and neoplasms, where it is believed that receptor dysregulation plays a role in oncogenic transformation and tumor progression. In addition to the ErbB1 holoreceptor, numerous studies demonstrate that cells synthesize soluble or secreted forms of ErbB1, i.e., sErbB1. Overexpression of ErbB1 in a variety of tumors has led us to hypothesize that sErbB levels also may be altered during oncogenesis, tumor progression, and/or metastasis; and that these molecules may be useful tumor biomarkers. To address this hypothesis we have developed an acridinium-linked immunosorbent assay (ALISA) specific for the extracellular domain of ErbB1 that can be used to quantify the levels of sErbB1 molecules in body fluids and conditioned culture media. This assay can also detect full-length ErbB1 in cell and tissue extracts. Our ALISA is characterized by high sensitivity (intra-assay LLD < 1 fmol/ml), a broad linear range (approximately 1 to 4000 fmol/ml), and good reproducibility (CVs < 10%). Specificity experiments show that this ALISA detects p170 ErbB1 and soluble forms of ErbB1 that embody extracellular subdomains I through IV, but not forms of sErbB1 lacking subdomain IV. Our ALISA does not detect full-length ErbB2, ErbB3, or ErbB4; or p105 soluble ErbB2. We report that serum sErbB1 levels of healthy women (median = 3716 fmol/ml), ranging in age from 43 to 76 years, differ significantly from those of healthy men (median = 24,512 fmol/ml), ranging in age from 25 to 79 years. Additional analyses do not indicate that serum sErbB1 levels change with age in either healthy men or women. Immunoprecipitation experiments show that monoclonal antibodies specific for extracellular epitopes of ErbB1 completely neutralize the detection of sErbB1 in normal human sera by ALISA. Finally, we show by immunoprecipitation and Western immunoblot analyses with monoclonal antibodies specific for the extracellular domain of ErbB1 that normal human female and male sera contain a approximately 110-kDa protein. We conclude that our ALISA is measuring the relative levels of this p110 sErbB1 analog in normal human sera. Our ALISA, therefore, should be useful for measuring the levels of ErbB1 and sErbB1 molecules in tumor biopsy specimens and body fluids, respectively, and for determining whether sErbB1, like ErbB1, is a useful tumor biomarker.

Generation and characterization of polyclonal antibodies specific for human p110 sEGFR.

The EGFR/ERBB family of receptor tyrosine kinases mediates intracellular signal transduction pathways important in the regulation of cell growth, differentiation, and transformation. We previously have reported the cloning and expression of a 3 kb alternative EGFR transcript which encodes a 110 kDa form of the receptor (p110 sEGFR). This receptor isoform is identical to the extracellular region of the full-length 170 kDa EGFR through amino acid 603; in addition, p110 sEGFR contains 78 unique carboxy-terminal amino acids. Here, we report the generation and characterization of polyclonal antisera specific for the unique carboxy-terminal sequence of p110 sEGFR. Polyclonal antisera were generated by immunizing rabbits with synthetic peptides corresponding to peptides contained within the unique carboxy-terminal sequence of p110 sEGFR. Immunoglobulin fractions from antisera which tested positive for immune reactivity to these peptides by ELISA were affinity-purified by protein G and peptide-based chromatography. This affinity-purified immunoglobulin fraction specifically recognizes p110 sEGFR by ELISA, immunoprecipitation, immunoblot analysis, and immunocytochemical methods. No cross-reactivity with full-length p170 EGFR is observed using any of these detection methods. These new polyclonal antibodies will be useful in determining the expression, localization, and function of p110 sEGFR, and importantly will allow us to distinguish between the expression of this receptor isoform and p170 EGFR.

Chapter 47 Centrin-Based Contractile Fibers: Chromatographic Purification of Centrin

Publisher Summary Centrin is a component of an assortment of calcium-modulated cytoskeletal fiber systems found associated with flagellar basal apparatus, centrosomes, and mitotic spindle poles of eukaryotic cells. It is an acidic, low-molecular-weight, calcium-binding phosphoprotein that belongs to the EF-hand superfamily of calcium-binding proteins centrin-based systems include striated flagellar roots, transition zone fibers, fibrous bundles, and pericentriolar lattice. Centrin-based fibers play a role in anchoring the flagellar basal apparatus to the nucleus, in positioning of basal bodies and centrioles during the cell cycle, in severing microtubules at the time of flagellar excision, and in coiling the paraflagellar rod of transverse flagella. In this chapter is presented detailed methods for generating milligram quantities of pure centrin. These methods involve expression of a cDNA clone encoding Chlamydomonas centrin in Escherichia coli, and chromatographic purification of the expressed protein. The chromatographic method described works well for the purification of centrin from isolated Chlamydomonas flagella. The chapter discusses the construction of E. coli BL21 pT7-5Cen, induction and lysis of E. coli BL21 pT7-5Cen, phenyl-sepharose CLdB chromatography, superose-12 gel filtration chromatography, mono Q anion-exchange chromatography, and the troubleshooting methods. Problems encountered include inactive ampicillin, which may result in the selection and growth of bacteria that have lost the pT7-Zen plasmid; failure of transcription induction because of inactive Isopropylthio- β -glactoside (IPTG) or T7 promoter; induction of protein products that are larger or smaller than centrin because of improper bacterial transcription and/or translation; inefficient digestion of the bacterial cell wall with lysozyme; and inefficient binding of centrin to a fouled phenylsepharose column. To troubleshoot and assess the efficiency of the purification procedure, it is advised to prepare SDS-PAGE samples of E. coli BL21 pT7-5Cen before and after IPTG induction, whole-cell lysate following sonication, P1, S1, P2, S2, column voids, and column elution fractions.