Identification and localization of a novel, cytoskeletal, centrosome-associated protein in PtK2 cells.

Antisera raised against centrin (Salisbury, J.L., A.T. Baron, B. Surek, and M. Melkonian. 1984. J. Cell Biol. 99:962-970) have been used, here, to identify a centrosome-associated protein with an Mr of 165,000. Immunocytochemistry indicates that this protein is a component of pericentriolar satellites, basal feet, and pericentriolar matrix of interphase cells. These components of pericentriolar material are, in part, composed of 3-8-nm-diam filaments, which interconnect to form a three-dimensional pericentriolar lattice. We conclude that the 165,000-Mr protein is immunologically related to centrin, and that it is a component of a novel centrosome-associated cytoskeletal filament system. Microtubule organizing centers such as the flagellar apparatus of algal cells, spindle pole body of yeast cells, and centrosome of mammalian cells are homologous structures essential for cytoplasmic organization and cellular proliferation. Molecular cloning studies have recently shown that the cell cycle gene product CDC31, required for spindle pole body duplication, shares 50% sequence homology with centrin (Huang, B., A. Mengersen, and V.D. Lee. 1988. J. Cell Biol. 107:133-140). The evolutionary conservation of centrin-related sequences and immunologic epitopes to microtubule organizing centers of divergent phylogeny suggests that a functional attribute(s) may have been conserved as well. Elucidation of a common thread between these related molecules may be fundamental to our understanding of cell structure and function.

The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells.

Monoclonal and polyclonal antibodies raised against algal centrin, a protein of algal striated flagellar roots, were used to characterize the occurrence and distribution of this protein in interphase and mitotic Chlamydomonas cells. Chlamydomonas centrin, as identified by Western immunoblot procedures, is a low molecular (20,000-Mr) acidic protein. Immunofluorescence and immunogold labeling demonstrates that centrin is a component of the distal fiber. In addition, centrin-based flagellar roots link the flagellar apparatus to the nucleus. Two major descending fibers extend from the basal bodies toward the nucleus; each descending fiber branches several times giving rise to 8-16 fimbria which surround and embrace the nucleus. Immunogold labeling indicates that these fimbria are juxtaposed to the outer nuclear envelope. Earlier studies have demonstrated that the centrin-based linkage between the flagellar apparatus and the nucleus is contractile, both in vitro and in living Chlamydomonas cells (Wright, R. L., J. Salisbury, and J. Jarvik. 1985. J. Cell Biol. 101:1903-1912; Salisbury, J. L., M. A. Sanders, and L. Harpst. 1987. J. Cell Biol. 105:1799-1805). Immunofluorescence studies show dramatic changes in distribution of the centrin-based system during mitosis that include a transient contraction at preprophase; division, separation, and re-extension during prophase; and a second transient contraction at the metaphase/anaphase boundary. These observations suggest a fundamental role for centrin in motile events during mitosis.

Serum sErbB1 and epidermal growth factor levels as tumor biomarkers in women with stage III or IV epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) has a high mortality rate, which is due primarily to the fact that early clinical symptoms are vague and nonspecific; hence, this disease often goes undetected and untreated until in its advanced stages. Sensitive and reliable methods for detecting earlier stages of EOC are, therefore, urgently needed. Epidermal growth factor (EGF) is a ligand for EGF receptor (ErbB1); this receptor is the product of the c-erbB1 proto-oncogene. ErbB1 overexpression is common in human ovarian carcinoma-derived cell lines and tumors, in which overexpression is thought to play a critical role in tumor etiology and progression. Furthermore, ErbB1 overexpression is associated with disease recurrence and decreased patient survival. Recently, we have developed an acridinium-linked immunosorbent assay that detects a approximately 110-kDa soluble analogue of ErbB1, ie., sErbB1, in serum samples from healthy men and women (A. T. Baron, et al., J. Immunol. Methods, 219: 23-43, 1998). Here, we demonstrate that serum p110 sErbB1 levels are significantly lower in EOC patients with stage III or IV disease prior to (P < 0.0001) and shortly after (P < 0.0001) cytoreductive staging laparotomy than in healthy women of similar ages, whereas EGF levels are significantly higher than those of age-matched healthy women only in serum samples collected shortly after tumor debulking surgery (P < 0.0001). We observe that the preoperative serum sErbB1 concentration range of advanced stage EOC patients barely overlaps with the serum sErbB1 concentration range of healthy women. In addition, we show that serum sErbB1 and EGF levels changed temporally for some EOC patients who were surgically debulked of tumor and who provided a second serum sample during the course of combination chemotherapy. Finally, we observe a significant positive association between sErbB1 and EGF levels only in serum samples of EOC patients collected prior to cytoreductive surgery (correlation coefficient = 0.61968; P = 0.0027). These data suggest that epithelial ovarian tumors concomitantly affect serum sErbB1 and EGF levels. In conclusion, these data indicate that serum sErbB1 and EGF (postoperative only) levels are significantly different between EOC patients and healthy women and that altered and/or changing serum sErbB1 and EGF levels may provide important diagnostic and/or prognostic information useful for the management of patients with EOC.

A preliminary study of serum concentrations of soluble epidermal growth factor receptor (sErbB1), gonadotropins, and steroid hormones in healthy men and women.

Soluble ErbB (sErbB) growth factor receptors are being investigated as cancer biomarkers. Gonadotropic and steroid hormones have been shown to modulate the expression of ERBB family members in vivo. Accordingly, the range of sErbB1 values and their relationship to gonadotropic and steroid hormones need to be established in healthy subjects to provide a baseline for future clinical studies. We assayed sera from healthy men and women to determine p110 sErbB1 concentrations by acridinium-linked immunosorbent assay (ALISA). Follicle-stimulating hormone (FSH), estradiol, and testosterone concentrations were measured using the ACS:180 Immunoassay Analyzer. Luteinizing hormone (LH) and progesterone concentrations were quantified using the Access Immunoassay System. Unadjusted for age, p110 sErbB1 concentrations in healthy men and women do not differ significantly. However, sErbB1 concentrations show a strong age-gender interaction, increasing with age in men but decreasing with age in women. Consequently, sErbB1 concentrations are significantly higher in premenopausal women compared with either postmenopausal women or age-matched men and in age-matched men compared with postmenopausal women. Serum sErbB1 concentrations show significant negative associations with both FSH and LH concentrations in healthy women and a significant positive association with FSH concentrations in healthy men. Univariate linear regression models show that these respective gonadotropic hormones and age are independent predictors of sErbB1 concentrations in men and women. Multivariate models show that when age and FSH and LH concentrations are mutually adjusted for each other, they account for 22% of the variability observed in sErbB1 concentrations in healthy women. These data support the hypothesis that gonadotropic and steroid hormones may modulate ERBB1 expression in vivo and suggest that age- and gonadotropin-adjusted sErbB1 concentrations may be of clinical utility. Furthermore, these data demonstrate that gender, age, menstrual cycle phase, menopausal status, and exogenous hormone use must be considered when using serum p110 sErbB1 concentrations as cancer biomarkers.

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.

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.

Monoclonal antibodies specific for peptide epitopes of the epidermal growth factor receptor's extracellular domain.

The ErbB tyrosine kinase receptor family plays an important role in normal cellular growth and differentiation. In addition, ErbB receptor family members are commonly amplified and overexpressed in various human neoplasms and tumor-derived cell lines, where it is believed that increased signalling as a result of receptor overexpression may play an important role in oncogenesis. Consequently, ErbB receptor family members are being investigated rigorously as potential biomarkers of cancer and as therapeutic targets in malignant tissues. Numerous studies now demonstrate the existence of "soluble" ErbB (sErbB) analogs in normal and cancerous tissues. These sErbB proteins embody the extracellular domain (ECD) of the receptor only; they are generated by either proteolytic cleavage or from truncated, alternatively spliced mRNA transcripts. Recently, we have identified an alternate transcript of the human c-erbB1 (Epidermal Growth Factor Receptor) proto-oncogene from placenta that encodes a sErbB1 protein of 60-kDa. This protein, p60 sErbB1, is glycosylated and secreted when expressed in transfected tissue culture cells in vitro. Although "soluble" receptor analogs may play important physiological roles in intercellular communication, tissue morphogenesis, tissue regeneration and repair, and embryogenesis by inhibiting or stimulating specific mitogenic and pattern forming signals, their mechanism of action has not been thoroughly elucidated. To further characterize sErbB1 expression in human tissues and cell lines and to better understand their role in carcinogenesis and normal development, we have generated monoclonal antibodies (MAbs) toward specific peptide epitopes of ErbB1 extracellular subdomains III and IV. These antibody reagents are described here and should be useful experimental, preparative, analytical, diagnostic, and therapeutic reagents for the study of sErbB1 molecules in normal development and cancer.

Localization of the centrin-related 165,000-Mr protein of PtK2 cells during the cell cycle.

In this study, we follow changes in localization of the centrin-related 165,000-Mr protein of PtK2 cells during the cell cycle. This protein is a component of a pericentriolar lattice that consists of pericentriolar satellites, pericentriolar matrix, and basal feet (Baron A.T., and J.L. Salisbury, J. Cell Biol. 107:2669-2678, 1988). By immunofluorescence microscopy, the 165,000-Mr protein is seen as a constellation of pericentrosomal spots. We observe that cells in late G1 and S are characterized by a dense centrosomal focus of spots with additional spots dispersed throughout the cytoplasm. In G2, one bright centrosomal focus of clustered spots is observed. As the cells proceed through prophase this single focus divides, forming two foci that move toward opposite sides of the nucleus. During prometaphase, each polar focus of spots disperses. At metaphase, the spots are distributed throughout each half-cytoplast from the poles to the chromosomes. During anaphase chromosome movement, some spots are seen beside and behind the trailing chromosome arms while others are clustered at the poles. At telophase, pericentrosomal spots radiate from the poles to surround each mass of chromatin. In early G1, pericentrosomal spots surround each newly formed nucleus. We conclude that the 165,000-Mr protein is a dynamic component of both the centrosome (pericentriolar matrix) and the mitotic apparatus (spindle matrix).

The pericentriolar lattice of PtK2 cells exhibits temperature and calcium-modulated behavior.

In this study, we demonstrate that manipulations of temperature and free calcium alter the morphology of the centrin-containing pericentriolar lattice of PtK2 cells. Immunofluorescence microscopy reveals that low-temperature incubation (4 degrees C) causes anti-centrin-labeled pericentrosomal spots to coalesce in the peripheral cytoplasm, and fuses small spots into larger spots near the cell center. At electron microscopic resolution, well-formed pericentriolar satellites appear around the centrioles in response to incubation at 4 degrees C. Elevated free calcium enhances these low-temperature-dependent effects. The data suggest that pericentrosomal spots correspond to one or more pericentriolar satellites, and that pericentriolar satellites and centrosomal matrix are interconvertable forms of the same material. Transient elevation of intracellular free calcium at 37 degrees C from a basal level of 3.7 x 10(-8) M to a peak level of 2.0 x 10(-7) M within 30 seconds with ionomycin results in a 35% increase in pericentrosomal spot number throughout the cytoplasm. The number of pericentrosomal spots is 50% larger 2 minutes after ionomycin addition; these spots are also nearer to the cell center as compared to 30 seconds after ionomycin addition. As intracellular free calcium returns to a basal level over 5 minutes, the number of spots and their cellular distribution resume a pretreatment value and pattern. We interpret these observations to indicate movement of pericentrosomal spots toward the cell center in response to the flux in intracellular free calcium. Alternatively, it is possible that no movement has occurred, but that the rise in free calcium has unmasked an epitope responsive to our anti-centrin antiserum. Regardless of the interpretation, we conclude that the pericentriolar lattice exhibits calcium-modulated behavior.

Centrin is a component of the pericentriolar lattice.

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 (M(r) 18,500-21,000), 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 M(r) 165,000; QT6 cells also contain centrin-related proteins (M(r) 64,000-165,000). We conclude that centrin is a component of the pericentriolar lattice of higher eukaryotic centrosomes.

Membrane IgM: interactions with the cortical cytoskeleton in the human lymphoblastoid cell line WiL2.

Cell-surface IgM (antigen receptor) sediments with the membrane fraction following osmotic lysis and homogenization of cells of the human lymphoblastoid cell line WiL2. In nonreducing buffers, SDS PAGE analysis of membrane pellets demonstrates that "native" membrane IgM exists as a dimer. In contrast to osmotic lysis, lysis of cells with the nonionic detergent Triton X-100 releases approximately 90% of the membrane-bound IgM into the supernatant; approximately 10% of the IgM pellets with the cytoskeletal fraction on centrifugation. Ligand challenge with either mu-chain-specific antibodies or concanavalin A induces a change in the state of membrane IgM making it refractory to detergent extraction, such that 43% of the IgM pellets during centrifugation. This ligand-induced retention of IgM is significantly diminished by the microfilament-disrupting agent cytochalasin D, whereas pretreatment of cells with sodium azide or colchicine results in no significant change in the percentage of membrane IgM retained by Triton X-100 residues. These results indicate that retention of IgM involves an association with the cortical actin-based cytoskeleton. Investigation of the structural basis for ligand-induced Triton X-100 retention of membrane IgM by using ferritin-conjugated antibodies, myosin subfragment S1, and stereo-imaging electron microscopy has revealed linkages between ligand-receptor (antigen-IgM) complexes and elements of the cortical actin-based cytoskeleton.

Calcium-modulated contractile proteins associated with the eucaryotic centrosome.

Affinity-purified antibodies that recognize the 20,000-dalton molecular weight (20 kd) striated flagellar root protein of Tetraselmis striata have been used to identify antigenic homologs in other eucaryotic organisms of diverse evolutionary origins. Among the green algae, Tetraselmis and Chlamydomonas, and their colorless relative, Polytomella, the 20-kd homologs appear associated with basal bodies. This occurs most prominently in the form of flagellar roots of both striated and microtubule subtended types. Among cultured mammalian cells (PtK2 and primary mouse macrophage cell lines), flagellar root protein homologs appear as basal feet, pericentriolar fibrils, and pericentriolar satellites. Mammalian sperm cells also show flagellar root protein homologs associated with their basal bodies. We envisage a functional role for these fibrous calcium-sensitive contractile proteins in altering the orientation of centrioles or basal bodies with their associated MTOCs by responding to topological calcium fluxes.

A randomized phase II study of sequential docetaxel and doxorubicin/cyclophosphamide in patients with metastatic breast cancer.

BACKGROUND: Docetaxel has yielded promising response rates as a component of doxorubicin-based combination schedules in patients with metastatic breast cancer, including docetaxel/doxorubicin and docetaxel/doxorubicin/cyclophosphamide (AC). This randomized two-stage phase II study was conducted to evaluate sequential treatment with docetaxel and AC as first-line treatment in patients with recurrent or metastatic breast cancer previously untreated with chemotherapy for metastatic disease. PATIENTS AND METHODS: Thirty-three patients were randomized to either docetaxel (100 mg/m(2)) on day 1 of a 21-day cycle for three cycles followed by AC (60/600 mg/m(2)) on day 1 of a 21-day cycle for three cycles (n = 17) or vice-versa (n = 16), without prophylactic granulocyte colony-stimulating factor support. In addition, we compared pre-treatment serum sErbB1 and sErbB2 protein concentrations with that of an age- and menopausal status-matched group of healthy women, and examined changes in serum sErbB1 and sErbB2 protein concentrations in these two treatment schedules. Data from each one of the two arms of the trial (docetaxel then AC, or AC and then docetaxel) were analyzed separately. RESULTS: Enrollment was suspended after the first-stage of accrual, based on statistical design. Confirmed objective response rates after six cycles of treatment were 35% [95% confidence interval (CI) 14% to 62%] with docetaxel then AC and 38% (95% CI 15% to 65%) with AC then docetaxel. Dose reductions were frequent and mostly due to grade 4 neutropenia. Median survival time was 2.5 years in the docetaxel then AC group, and 1.1 years in the AC then docetaxel group. Serum sErbB1 concentrations were not significantly different between the study patients and healthy women, and did not change significantly after three and six cycles of treatment. In contrast, serum sErbB2 concentrations were significantly higher in the study patients compared with healthy women and tended to decrease after three and six cycles of treatment. CONCLUSIONS: Response rates at the end of six cycles of treatment, which led to termination of accrual after the first stage using either the sequence of docetaxel first or docetaxel after AC chemotherapy, were lower than anticipated. However, median survival times and median progression-free survival times are similar to those reported in other studies. These data further suggest that additional studies to assess whether serum sErbB2 concentrations are useful predictors of responsiveness to chemotherapy are warranted.