Blocking HER-2/HER-3 function with a dominant negative form of HER-3 in cells stimulated by heregulin and in breast cancer cells with HER-2 gene amplification.

Amplification and overexpression of the HER-2 (neu/ erbB-2) gene in human breast cancer are clearly important events that lead to the transformation of mammary epithelial cells in approximately one-third of breast cancer patients. Heterodimer interactions between HER-2 and HER-3 (erbB-3) are activated by neu differentiation factor/heregulin (HRG), and HER-2/HER-3 heterodimers are constitutively activated in breast cancer cells with HER-2 gene amplification. This indicates that inhibition of HER-2/HER-3 heterodimer function may be an especially effective and unique strategy for blocking the HER-2-mediated transformation of breast cancer cells. Therefore, we constructed a bicistronic retroviral expression vector (pCMV-dn3) containing a dominant negative form of HER-3 in which most of the cytoplasmic domain was removed for introduction into cells. By using a bicistronic retroviral vector in which the antibiotic resistance gene and the gene of interest are driven by a single promoter, we attained 100% coordinate coexpression of antibiotic resistance with the gene of interest in target cell populations. Breast carcinoma cells with HER-2 gene amplification (21 MT-1 cells) and normal mammary epithelial cells without HER-2 gene amplification from the same patient (H16N-2 cells) were infected with pCMV-dn3 and assessed for HER-2/ HER-3 receptor tyrosine phosphorylation, p85PI 3-kinase and SHC protein activation, growth factor-dependent and -independent proliferation, and transformed growth in culture. Dominant negative HER-3 inhibited the HRG-induced activation of HER-2/HER-3 and signaling in H16N-2 and 21 MT-1 cells as well as the constitutive activation of HER-2/HER-3 and signaling in 21 MT-1 cells. Responses to exogenous HRG were strongly inhibited by dominant negative HER-3. In contrast, the proliferation of cells stimulated by epidermal growth factor was not apparently affected by dominant negative HER-3. The growth factor-independent proliferation and transformed growth of 21 MT-1 cells were also strongly inhibited by dominant negative HER-3 in anchorage-dependent and independent growth assays in culture. Furthermore, the HRG-induced or growth factor-independent proliferation of 21 MT-1 cells was inhibited by dominant negative HER-3, whereas the epidermal growth factor-induced proliferation of these cells was not: this indicates that dominant negative HER-3 preferentially inhibits proliferation induced by HER-2/HER-3.

Detection of multiple fibronectin isoforms in fetal fibroblast monolayer culture: a novel method for the qualitative and quantitative detection of multiple antigens.

Analyzing the expression of multiple distinct antigens within a single monolayer culture involves cumbersome immunostaining techniques. We describe a simple and economical procedure for the detection and quantification of multiple antigens within a single monolayer. By generating an immunohistochemical grid which divides a monolayer in a standard tissue culture dish into 20 distinct areas, we were able to detect and quantify four individual fibronectin (FN) isoforms within a single fibroblast monolayer culture. Quantification of each isoform was performed using a modified enzyme-linked immunoassay. In addition, within the same monolayer, each FN isoform was detected using standard immunohistochemical detection with DAB visualization. Using this novel approach to immunohistochemical analysis we determined that within the first 4 days of culture, the quantity of all FN isoforms increases faster than the number of cells. However, upon reaching confluency, the quantity of FN/cell drops dramatically. After reaching confluency, the amount of FN/cell levels off and remains constant within the postconfluent monolayer. Statistical analysis of the quantity of FN/cell indicates that a significant reduction in the amount of FN/cell occurs in the 2 days prior to reaching confluency. The distribution of all the FN isoforms, with the exception of B-FN, was found along the length of the cell body. In contrast, the distribution of B-FN was altered in postconfluent monolayers where it was detected only in distinct locations within the monolayer.

Expression and assembly of the angiogenic marker B-fibronectin by endothelial cells in vitro: regulation by confluency.

Recent studies have demonstrated the association of the rare fibronectin (FN) isoform B-FN with newly formed blood vessels. Although B-FN is a unique angiogenesis marker, it is not clear whether B-FN associated with the neovasculature is expressed by the endothelial cells (EC) or obtained from the microenvironment. Here we report on a study analyzing the expression and assembly of B-FN by bovine microvascular EC (BMEC) in vitro. We determined that BMEC express B-FN and assemble it into fibrils during monolayer culture. Furthermore, in addition to assembling endogenous B-FN, subconfluent BMEC can assemble exogenous B-FN provided by a non-EC source. Upon reaching confluency the assembly and expression of B-FN by BMEC is inhibited and the previously assembled B-FN is eventually eliminated from postconfluent EC monolayers. Indeed, confluent BMEC assemble neither endogenous nor exogenous B-FN, while they continue to assemble other FN isoforms. We conclude that BMEC in vitro express B-FN and assemble B-FN fibrils using endogenous as well as exogenous B-FN. The expression and assembly of B-FN are tightly regulated by confluency. Our observations in vitro are a plausible explanation for the absence of B-FN in established blood vessels in vivo, and the subsequent reappearance of B-FN in angiogenic vessels. Furthermore, since our observations of B-FN assembly in BMEC monolayers correspond to previously published observations in vivo, B-FN may be utilized as an appropriate marker for angiogenic behavior of EC in vitro.

Basic fibroblast growth factor receptors and their prognostic value in human breast cancer.

We performed a saturation binding study with 125I-labeled FGF (fibroblast growth factor)-2 in a nonselected series of 250 human primary breast cancers. Two hundred twenty-five breast cancer biopsies possessed bFGFR (basic FGF receptor). The median dissociation constant was 0.35 nM (range, 0.014-1.9), and the median concentration was 1126 fmol/mg protein (range, 49-7328). FGFR-1 was localized, using a specific monoclonal antibody, in cancerous cells and in epithelial cells in normal breast or in benign tumors. In all of the tissues studied, light stromal cell staining was also observed. Thus, the localization of FGFR-1 in carcinoma cells supports the hypothesis that an important part of FGF-2 binding reflects binding to FGFR-1. bFGFR concentrations were positively correlated to estrogen receptor and progesterone receptor levels. Cox univariate analyses showed that the bFGFR (> or = upper quartile) was associated to longer relapse-free survival [P = 0.004; RR (risk ratio), 0.46] and overall survival (P = 0.001; RR, 0.35); age, estrogen receptor levels, progesterone receptor levels, node involvement, tumor diameter, and histoprognostic grading were prognostic, also. In Cox multivariate analyses, only the bFGFR, age, node involvement, and histoprognostic grading were prognostic factors; the bFGFR was associated with longer relapse-free survival (P = 0.03; RR, 0.4) and overall survival (P = 0.009; RR, 0.3). The present study confirms that FGF could be an important regulator of human breast cancer growth and that patients with a high level of bFGFR had a better prognosis.

Efficient purification of mouse anti-FGF receptor IgM monoclonal antibody by magnetic beads.

Affinity chromatography has been widely used for the purification of monoclonal antibodies (MAb). Traditionally, activated agarose beads conjugated with specific antisera have been used as a solid support in chromatographic protein purification. Magnetic beads conjugated with various antibodies have recently become an alternative method for the isolation of diverse proteins, nucleic acids, and cell types. In this study, murine anti-fibroblast growth factor receptor 1 (FGFR1) immunoglobulin M (IgM) was isolated from protein solutions to compare immunoaffinity column chromatography and magnetic bead IgM purification methods. Using immobilized rat anti-mouse IgM MAb, an UltraLink 1-ethyl-3-(3-dimethylaminopropyl)carboiimide (EDC)/diaminodipropylamine (DADPA) immunoaffinity column and polystyrene-coated magnetic beads were used for the purification of mouse IgM from bovine serum albumin/phosphate-buffered saline (BSA/PBS) as well as from crude ascites. Protein quantitation and percent IgM yield were determined by reducing SDS-PAGE electrophoresis followed by silver staining, then IgM and protein contaminants were quantitated using densitometry analysis. IgM anti-FGFR1 binding specificity and immunologic activity were determined by enzyme-linked immunosorbant assay (ELISA). This study demonstrates that magnetic bead isolation of IgM from ascites is more effective than traditional affinity chromatography purification as determined by greater IgM yield, purity, and immunologic activity.

Characterization of a functional angiotensin IV receptor on coronary microvascular endothelial cells.

A new class of angiotensin receptors has recently been identified that exhibits both high specificity and affinity for the hexapeptide (3-8) fragment of angiotensin II, angiotensin IV (AngIV). Here, utilizing radioligand binding, we fully characterize AngIV binding at the AT4 receptor on cultured bovine coronary venular endothelial cells (CVEC), and report that when AngIV and bFGF are presented simultaneously an enhancement of DNA synthesis results that is significantly greater than that produced by bFGF alone. The level of DNA synthesis was determined by the incorporation of [3H]thymidine into quiescent CVEC monolayers following exposure to 10 nM AngIV and 10 ng/ml bFGF for 1, 3, 5, 7, 9, or 11 days. A significant enhancement of DNA synthesis (P < 0.01) was seen following 3, 5, 7, 9 and 11 days exposure. In addition, AngIV does not bind to bFGF or heparin, and conversely, bFGF is unable to compete for AngIV binding which suggests that this synergistic response is mediated by independent receptors for these ligands. Results of this study indicate that microvascular endothelial cells are significantly more responsive to bFGF in the presence of nanomolar concentrations of AngIV.

Differential growth factor production, secretion, and response by high and low metastatic variants of B16BL6 melanoma.

Low levels of tyrosine and phenylalanine alter the metastatic phenotype of B16BL6 murine melanoma. In this study, we investigated expression and secretion of fibroblast growth factor-like (FGF-like) and transforming growth factor beta-like (TGF beta-like) molecules as well as the biological effect of basic FGF (bFGF) and TGF beta 1 on high (NDP) and low (LTP) metastatic variants of B16BL6 melanoma. Both NDP and LTP cells expressed bFGF-like and TGF beta-like polypeptides as detected by Western blot analysis. An M(r) 29,000 bFGF-like form eluted from heparin-Sepharose by 0.6 M NaCl was found in extracts of both NDP and LTP cells. Elution at 0.6 M NaCl suggested that this M(r) 29,000 form might be more closely related to FGF-5 than to bFGF. In addition, cell extracts of LTP, but not NDP cells, contained an M(r) 47,000 monomeric bFGF-like form that was not retained on heparin-Sepharose. Three major specific immunoreactive forms of M(r) 44,000, 36,000, and 29,000 were present in conditioned medium from NDP cells. The M(r) 29,000 form present in the conditioned medium of NDP cells was retained on heparin-Sepharose. Only the M(r) 44,000 and 36,000 FGF-like molecules were detected in conditioned medium from LTP cells, and they were also not retained on heparin-Sepharose. Anti-TGF beta antibody that recognized both TGF beta 1 and TGF beta 2 detected 3 different TGF beta-like forms (M(r) 25,000, 23,000 and 22,000) in NDP and LTP cell extracts. Conditioned medium from NDP cells contained an M(r) 38,000 form of TGF beta; however, no immunoreactive forms were found in conditioned medium from LTP cells. Thus, the NDP-LTP differences in this melanoma system were primarily in growth factor secretion, not expression. The effect of exogenous bFGF and TGF beta 1 on proliferation of LTP and NDP cells was determined by [methyl-3H]thymidine uptake. bFGF stimulated proliferation of NDP cells; whereas, LTP cells exhibited no increase in proliferation. Both NDP and LTP cells responded to TGF beta 1. Proliferation of NDP cells was inhibited more by this growth factor than was proliferation of LTP cells. When NDP and LTP cells were incubated with 5 ng/ml TGF beta 1 and various amounts of bFGF, the effect of TGF beta 1 was masked. Antibody depletion of bFGF-like molecules from NDP conditioned medium resulted in the decreased proliferation of NDP cells but not LTP cells. Depletion of TGF beta-like molecules resulted in increased proliferation of LTP cells but did not affect NDP cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Basic fibroblast growth factor is a testicular germ cell product which may regulate Sertoli cell function.

Previously, a proteinacious factor secreted by a mixture of rat testicular spermatocytes and round spermatids was shown to stimulate transferrin mRNA and protein levels in Sertoli cells. To identify the germ cell-secreted proteins which affect Sertoli cell functions, concentrated germ cell-conditioned medium was fractionated by reverse-phase HPLC. The fraction which eluted at 35% acetonitrile increased transferrin secretion in Sertoli cell cultures 2.4-fold above the basal level. Both the active fraction and a protein extract from cultured germ cells were positive for basic fibroblast growth factor (bFGF) as determined by Western blot analysis and immunoprecipitation. The apparent molecular sizes of the immunoreactive proteins were 30, 27, and 24 kilodaltons (kDa). By immunohistochemistry, bFGF was shown to be present in pachytene spermatocytes and Leydig cells. The bFGF receptor was also examined by immunohistochemistry and found to be present in Leydig cells, round and elongated spermatids, and Sertoli cells. The presence of receptors was more pronounced in stages I-VIII. Western blot analysis confirmed that the receptors were expressed in isolated round spermatids, elongated spermatids, and Sertoli cells. Two major receptor species with apparent molecular sizes of 120 and 145 kDa were detected in the rat testis. Germ cells contained both of these receptors, but Sertoli cells possessed only the 120-kDa receptor. From these experiments, it is evident that bFGF is a germ cell product which may regulate Sertoli cell function. The expression of bFGF and its receptor may be an important component of germ cell-Sertoli cell and/or germ cell-germ cell communication during spermatogenesis.

Anoxia-reoxygenation-induced, neutrophil-mediated endothelial cell injury: role of elastase.

The aim of this study was to assess the role of neutrophilic elastase in anoxia-reoxygenation-induced, neutrophil-mediated injury to microvascular endothelium. Cultured bovine microvascular endothelial cells were grown to confluence and labeled with 51Cr. The endothelial cells were exposed to a 30-min period of anoxia and subsequently reoxygenated. Endothelial cell injury, quantitated as 51Cr release and cell detachment, was determined 8 h after reoxygenation. Addition of neutrophils upon reoxygenation enhanced the anoxia-reoxygenation-induced increase in 51Cr release and cell detachment. The neutrophil-mediated injury was associated with elastase release from the neutrophils. Four agents were used to inhibit neutrophilic elastase activity: Eglin C, methoxysuccunyl-Ala2-Pro-Val-CH2Cl, L658,758, and a monoclonal antibody against neutrophilic elastase. All elastase inhibitors attenuated the neutrophil-mediated endothelial cell detachment but not 51Cr release. Addition of purified human neutrophilic elastase, at a level that mimicked the release from neutrophils, increased cell detachment in endothelial cells exposed to anoxia-reoxygenation but did not affect 51Cr release. Our results indicate that elastase plays an important role in anoxia-reoxygenation-induced, neutrophil-mediated endothelial cell dysfunction.

An in vitro model of ischemia/reperfusion-induced microvascular injury.

The major objective of this study was to develop an in vitro model of ischemia/reperfusion (I/R)-induced microvascular injury. Cultured venular endothelial cells were grown to confluency, labeled with 51Cr, and exposed to different durations of anoxia (0.5, 1, 2, 3, and 4 h). 51Cr release and cell detachment (indexes of cell injury) were determined at different times after reoxygenation (1, 2, 4, 6, 8, and 18 h). Because in vivo studies have implicated neutrophils in I/R injury, in some experiments human neutrophils were added to the endothelial cells upon reoxygenation. Periods of anoxia greater than or equal to 2 h resulted in 70-80% 51Cr release and 80-95% cell detachment upon reoxygenation. Under these conditions (near maximal injury), the addition of neutrophils produced negligible effects. Periods of anoxia less than or equal to 1 h resulted in 30-40% 51Cr release and 50-60% cell detachment. Under these conditions (moderate cell injury), addition of neutrophils enhanced endothelial cell injury. Using a 30-min period of anoxia, we also assessed the effects of superoxide dismutase (SOD; 300 U/ml) and allopurinol (20 microM) on anoxia/reoxygenation (A/R)-induced injury in the presence or absence of neutrophils. In the absence of neutrophils, SOD or allopurinol did not protect against A/R-induced injury. However, in the presence of neutrophils, both SOD and allopurinol attenuated the increases in 51Cr release. The results derived using this in vitro model of I/R injury are largely consistent with published in vivo studies. Thus this in vitro model may provide further insights regarding the mechanisms involved in I/R injury.

Superoxide mediates reperfusion-induced leukocyte-endothelial cell interactions.

The objective of this study was to determine whether superoxide mediates the leukocyte-endothelial cell interactions elicited by reperfusion (reoxygenation) of ischemic (hypoxic) tissues. Mesenteric and intestinal blood flows were reduced to 20% of control for 1 h, followed by 1 h of reperfusion. Sixty minutes after reperfusion, red blood cell velocity (Vr), leukocyte rolling velocity (Vw), and the number of adherent leukocytes were measured in mesenteric venules. Then, either human superoxide dismutase (hSOD), hydrogen peroxide-inactivated hSOD, or MoAb IB4 (a monoclonal antibody against the leukocyte adhesion molecule CD18) was injected intravenously. Ten minutes later, repeat measurements were obtained and compared with pretreatment values. hSOD attenuated reperfusion-induced neutrophil adherence and increased Vw/Vr, an index of the fracture stress between leukocytes and endothelium. Peroxide-inactivated hSOD did not alter any parameter. MoAb IB4 attenuated reperfusion-induced adherence but did not alter Vw/Vr. In a correlate study, cultured bovine microvascular endothelium was exposed to 30 min of anoxia, followed by 60 min of reoxygenation. Cat neutrophils were added during reoxygenation. Reoxygenation-induced leukocyte adherence was attenuated by either hSOD or MoAb IB4 but not by inactivated hSOD. Adherence of phorbol 12-myristate 13-acetate-activated cat neutrophils to plastic was unaffected by hSOD or inactive hSOD, yet MoAb IB4 virtually abolished the response. These results indicate that superoxide mediates reperfusion-induced leukocyte adherence and that endothelial cells are required for this superoxide-mediated adherence.

Physiology and pathobiology of the microcirculation.

The delivery of nutrients to the tissues and the removal of waste products from the tissues is made possible by forcing a stream of blood through an arborizing network of microscopic blood vessels that comprise the microcirculation. The rapidity of the flow stream and, therefore, the rate of nutrient delivery to the tissue, is regulated by the automatic adjustment of the caliber of the precapillary arterioles that serve as the primary loci of vascular resistance. Exchange between the blood stream and the parenchymal cells occurs in capillaries and pericytic venules. Pathologic processes such as inflammation, diabetes, ischemia, and hypertension are characterized by abnormalities in microvascular structure and function.

Adenosine and hypoxia stimulate proliferation and migration of endothelial cells.

The proliferation of bovine aortic or coronary venular endothelial cells (EC) in vitro was stimulated by the addition of adenosine (0.5 or 5.0 microM) to the culture medium. Cell counts of adenosine-treated aortic EC were 23-76% and coronary venular EC 19-52% greater than nontreated controls. Because adenosine is known to be released by hypoxic tissues, cell proliferation was quantitated when aortic EC were grown at 2% O2. Cell counts were 41-102% greater under hypoxic conditions than when cells were grown at standard tissue culture conditions (approximately 20% O2). When culture medium conditioned by coronary EC grown at 2% O2 was added to EC growing at standard conditions, cell counts were 24-69% greater than controls with medium conditioned by coronary EC grown at 20% O2. This suggests that hypoxia causes endothelial cells to release a factor(s) into the medium that can stimulate cell proliferation. The addition of the adenosine receptor blocker 8-phenyltheophylline (10(-5) M) prevented the stimulation of proliferation caused by hypoxia-conditioned medium, 2% O2 or 5.0 microM adenosine, suggesting that adenosine mediates its effect via an external membrane receptor. Adenosine also stimulated EC chemotaxis. Taken together, these results suggest that adenosine, released as a result of tissue hypoxia, may act as an angiogenic stimulus for the growth of new blood vessels.

The nucleolar RNA-binding protein B-36 is highly conserved among plants.

The nucleolar protein B-36 is an RNA-associated protein which has a number of properties in common with pre-mRNA-binding proteins (hnRNP proteins). Like the hnRNP proteins, B-36 appears to be evolutionarily conserved among various eukaryotes (protists and several animal species). The conservation of B-36 throughout the plant kingdom has been investigated using a panel of nine monoclonal antibodies previously shown to recognize a minimum of four different epitopes in Physarum B-36, the protein used to generate the monoclonal antibodies. Two of the epitopes (I and III) are widely conserved in 34 kDa proteins (presumably B-36 homologues) from the various species tested (Chlamydomonas, moss, fern, oat, onion, carrot, and bean). Using immunofluorescence localization in moss and carrot protoplasts, the cross-reacting proteins were shown to be restricted to the nucleolus, further confirming their probable homology to B-36. Epitopes I and III are also unique to the B-36 homologues as demonstrated by the failure of any other bands to cross-react. Another epitope (IV) was specifically recognized in the plant B-36 homologues but exhibited greatly reduced affinity for the monoclonal antibody relative to Physarum B-36. The remaining epitope (II), unlike the others, exhibited variable conservation in the plant B-36 homologues and, in addition, was present in several other seemingly unrelated proteins. Finally, several of the plant species exhibited two cross-reacting variants at roughly the 34 kDa position and in at least one of these cases a single monoclonal antibody was able to distinguish between the two variants, a result indicating that the variants do have bona fide structural differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Venular endothelial cells from bovine heart.

Coronary venular endothelial cells were isolated by a bead-perfusion technique that allowed the selection of endothelial cells from venules of a specific size. Culture conditions for the microvascular cells were established. Cells grew well in supplemented Dulbecco's modified Eagle's medium. The effect of various substrata on the proliferation of the venular endothelial cells was determined. Matrigel, gelatin, and fibronectin supported high levels of proliferation. Cell shape was correlated with ability of the substratum to support cell proliferation. Cells exhibiting a broad, flattened morphology achieved high levels of proliferation. The formation of vessel meshworks by the coronary venular endothelial cells provides an in vitro model for the study of coronary angiogenesis. Confluent monolayers of these cells can be utilized to examine mechanisms of water and protein transport across coronary venules.

Immunochemical comparison of peptide angiogenic factors.

A panel of 40 monoclonal antibodies was constructed in response to cationic endothelial cell growth factor (c-ECGF), the cationic peptide mitogen isolated from endothelial mitogen. The monoclonal antibodies were assayed by dot blot for immunoreactivity to various other peptide angiogenic factors. The panel of monoclonal antibodies to c-ECGF exhibited complete cross-reactivity with pituitary fibroblast growth factor and sarcoma-derived growth factor. A group of 28 monoclonal antibodies was found to exhibit reactivity to anionic endothelial mitogen (a-ECGF), brain fibroblast growth factor, endothelial cell growth factor, and retina-derived growth factor. None of the monoclonal antibodies was found to react with epidermal growth factor or platelet-derived growth factor. These data provide an immunological basis for grouping heparin-binding endothelial cell growth factors into anionic and cationic groups.

Characterization of the nucleolar protein, B-36, using monoclonal antibodies.

A panel of nine monoclonal antibodies has been produced against a major nuclear protein, B-36, purified from the slime mold Physarum polycephalum. B-36, a 34 kD protein biochemically similar to the major structural proteins of mammalian hnRNP particles, was previously shown to be largely associated with the nucleolus. Eight of the monoclonal antibodies are specific for B-36 protein in Physarum and at least three different epitopes are represented among these eight. Using the monoclonal antibodies B-36 has been shown to be localized exclusively to the nucleolus in actively-growing Physarum cultures. The nucleolar localization of B-36 is dependent on the presence of intact RNA, but not DNA, supporting the hypothesis that B-36 is associated with nucleolar RNA, possibly in some analogous manner to the interaction of the related proteins within heterogeneous nuclear ribonucleoproteins (hnRNP) particles. B-36 is apparently a highly conserved nucleolar protein in eukaryotes as all eight of the monoclonal antibodies specific for B-36 in Physarum are also specific for a 34.5 kD nucleolar protein in rat liver. This indicates that a minimum of three distinct epitopes are conserved in B-36 protein from slime mold to rat.