Presence and indirect immunofluorescent localization of calmodulin in Paramecium tetraurelia.

In this paper we demonstrate the presence and localization of calmodulin, a calcium-dependent regulatory protein, in the ciliated protozoan Paramecium tetraurelia. Calmodulin is demonstrated by several criteria: (a) the ability of whole cell Paramecium extracts to stimulate mammalian phosphodiesterase activity, (b) the presence of an acidic, thermostable, 17,000-dalton polypeptide whose mobility shifts in SDS polyacrylamide gel electrophoresis in the presence of Ca2+, and (c) the affinity of antibodies against mammalian calmodulin for a Paramecium component as demonstrated by both indirect immunofluorescent localization and radioimmunoassay. Indirect immunofluorescence studies reveal that Paramecium calmodulin is distributed in three distinct regions of the cell, i.e., (a) large, spherical cytoplasmic organelles representing perhaps the food vacuoles or other vacuolar inclusions of the cell, (b) along the entire length of oral and somatic cilia, and (c) along a linear punctate pattern corresponding to the kinetics (basal bodies) of the cell.

An alternatively processed mRNA from the avian c-erbB gene encodes a soluble, truncated form of the receptor that can block ligand-dependent transformation.

At least four major transcripts are produced by the avian c-erbB/epidermal growth factor receptor gene. cDNAs corresponding to the smallest one, a 2.6-kb transcript, were isolated from an adult chicken liver cDNA library. Sequence analysis revealed that the 3' end of one cDNA clone diverged from the known sequence of the extracellular ligand-binding domain (LBD) of the full-length receptor. A genomic DNA subfragment that contained this unique 3' divergent end was isolated. Sequence analysis of this genomic DNA fragment revealed that the 2.6-kb c-erbB transcript is produced by alternative processing. Translation of this 2.6-kb transcript would produce a secreted, truncated receptor molecule which contains the amino-terminal three-fourths of the extracellular LBD of the native receptor. COS1 cells and primary chicken embryo fibroblast cells were transfected with expression vectors that contained the 2.6-kb c-erbB cDNA. Conditioned medium from these transfected cells contained a 70-kDa protein that was specifically immunoprecipitated by a polyclonal antiserum directed against the LBD of the avian c-erbB gene product. The 70-kDa truncated receptor could be coimmunoprecipitated from conditioned medium of transfected COS1 cells that was supplemented with recombinant human transforming growth factor alpha (TGF alpha) by a monoclonal antibody against human TGF alpha. Additionally, transfected chicken embryo fibroblast cells that overexpressed the 70-kDa truncated receptor were blocked in their ability to form TGF alpha-dependent colonies in soft agar. These data suggest that the secreted, truncated receptor encoded by the 2.6-kb c-erbB transcript can bind to TGF alpha and may play an important growth-regulatory function in vitro.

Differential modulation of plasminogen activator gene expression by oncogene-encoded protein tyrosine kinases.

Urokinase-type plasminogen activator (uPA) gene transcription is increased > or = 50-fold in chicken embryo fibroblasts (CEF) following transformation by the protein tyrosine kinase pp60v-src. Protein phosphorylation appears to play a critical role in uPA gene expression in these cells; protein kinase C-activating phorbol esters cooperate with pp60v-src to synergistically increase uPA mRNA, whereas cyclic AMP (cAMP)-dependent protein kinase-activating agents (e.g., 8-bromo cAMP) repress uPA mRNA levels. To explore the relationship between transforming oncogenes and uPA gene expression, uPA mRNA levels were measured in CEF infected with selected avian retroviruses. We report that v-ras and the transforming protein tyrosine kinases v-src, v-yes, and v-ros all increase cellular uPA mRNAs. However, transformation with the protein tyrosine kinase encoded by v-erbB, or the nuclear proteins encoded by v-jun, v-ski, or v-myc, did not increase uPA mRNA detectably. Ras and all of the protein tyrosine kinases analyzed, including the v-erbB product, but none of the nuclear oncoproteins sensitized cells to phorbol ester induction of uPA gene expression. Thus, increased uPA gene expression is not simply a secondary consequence of cell transformation but, rather, is regulated or comodulated by only a subset of oncogene products. Analysis of cells expressing site-directed mutants of pp60v-src showed that the induction of the uPA gene is dependent on protein tyrosine kinase catalytic activity, myristylation, and plasma membrane localization. However, these properties together are not sufficient; an additional feature in the src homology 2 domain is also required. The major sites of serine phosphorylation, serines 12 and 17, and the autophosphorylation site, tyrosine 416, are not essential for uPA gene induction. However, the reduction of uPA mRNA in pp60v-src-transformed cells by 8-bromo cAMP is dependent on tyrosine 416.

Proviral insertional activation of c-erbB: differential processing of the protein products arising from two alternate transcripts.

Proviral insertional activation of c-erbB results in the expression of two alternate transcripts (ENV+ and ENV-). We used cDNA clones representing the two alternate transcripts to generate stably transformed quail fibroblast cell lines which express the products of these transcripts independently. Analysis of the co- and posttranslational processing of the insertionally activated c-erbB products expressed in these cell lines revealed that the protein products of the ENV+ and ENV- transcripts were processed differently. The ENV+ transcript produced a primary translation product which was rapidly cotranslationally cleaved near the amino terminus to form a 79,000-Mr product. This protein product was efficiently converted to a higher-molecular-weight form, of between 82,000 and 88,000 (gp82-88), which was terminally glycosylated and expressed on the cell surface. A small portion of the ENV+ primary translation product underwent a second proteolytic cleavage to generate an unglycosylated 53,000-Mr species. In contrast, the primary translation product of the ENV- transcript, p80, was not proteolytically processed; this precursor form was rapidly converted to two discrete glycosylation intermediates, gp82 and go84. Only a small portion (less than 10%) of the total ENV- insertionally activated c-erbB product was slowly converted to the terminally glycosylated cell surface form, gp85-88. The processing differences that distinguished the ENV+ and ENV- products were similar to processing differences that we observed in parallel studies on the viral erbB products of the avian erythroblastosis viruses AEV-H and AEV-R, respectively. Since all four erbB protein products shared the same number, position, and sequence context of potential N-linked glycosylation sites, yet differed in the extent of their carbohydrate maturation, these data suggest that the mechanisms used by these truncated receptor molecules to associate with cellular membranes may be distinct.

Activation of c-fos gene expression by a kinase-deficient epidermal growth factor receptor.

The intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR) has been shown to be responsible for many of the pleiotropic intracellular effects resulting from ligand stimulation [W.S. Chen, C.S. Lazar, M. Poenie, R.Y. Tsien, G.N. Gill, and M.G. Rosenfeld, Nature (London) 328:820-823, 1987; A.M. Honegger, D. Szapary, A. Schmidt, R. Lyall, E. Van Obberghen, T.J. Dull, A. Ulrich, and J. Schlessinger, Mol. Cell. Biol. 7:4568-4571, 1987]. Recently, however, it has been shown that addition of ligand to cells expressing kinase-defective EGFR mutants can result in the phosphorylation of mitogen-activated protein kinase (R. Campos-González and J.R. Glenney, Jr., J. Biol. Chem. 267:14535-14538, 1992; E. Selva, D.L. Raden, and R.J. Davis, J. Biol. Chem. 268:2250-2254, 1993), as well as stimulation of DNA synthesis (K.J. Coker, J.V. Staros, and C.A. Guyer, Proc. Natl. Acad. Sci. USA 91:6967-6971, 1994). Moreover, mitogen-activated protein kinase has been shown to phosphorylate the transcription factor p62TCF in vitro, leading to enhanced ternary complex formation between p62TCF, p67SRF, and the c-fos serum response element (SRE) [H. Gille, A.D. Sharrocks, and P.E. Shaw, Nature (London) 358:414-417, 1992]. On the basis of these observations, we have investigated the possibility that the intrinsic tyrosine kinase activity of the EGFR may not be necessary for transcriptional activation mediated via p62TCF. Here, we demonstrate that a kinase-defective EGFR mutant can signal ligand-induced expression of c-fos protein and that a significant component of this induction appears to be mediated at the transcriptional level. Investigation of transcriptional activation mediated by the c-fos SRE shows that this response is impaired by mutations in the SRE which eliminate binding of p62(TCF). These data indicate that information inherent in the structure of the EGFR can be accessed by ligand stimulation independent of the receptor's catalytic kinase function.

Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells.

Amphiregulin is a heparin-binding epidermal growth factor (EGF)-related peptide that binds to the EGF receptor (EGF-R) with high affinity. In this study, we report a role for amphiregulin in androgen-stimulated regulation of prostate cancer cell growth. Androgen is known to enhance EGF-R expression in the androgen-sensitive LNCaP human prostate carcinoma cell line, and it has been suggested that androgenic stimuli may regulate proliferation, in part, through autocrine mechanisms involving the EGF-R. In this study, we demonstrate that LNCaP cells express amphiregulin mRNA and peptide and that this expression is elevated by androgenic stimulation. We also show that ligand-dependent EGF-R stimulation induces amphiregulin expression and that androgenic effects on amphiregulin synthesis are mediated through this EGF-R pathway. Parallel studies using the estrogen-responsive breast carcinoma cell line, MCF-7, suggest that regulation of amphiregulin by estrogen may also be mediated via an EGF-R pathway. In addition, heparin treatment of LNCaP cells inhibits androgen-stimulated cell growth further suggesting that amphiregulin can mediate androgen-stimulated LNCaP proliferation. Together, these results implicate an androgen-regulated autocrine loop composed of amphiregulin and its receptor in prostate cancer cell growth and suggest that the mechanism of steroid hormone regulation of amphiregulin synthesis may occur through androgen upregulation of the EGF-R and subsequent receptor-dependent pathways.

Metformin alters DNA methylation genome-wide via the H19/SAHH axis.

The molecular mechanisms underlying the antineoplastic properties of metformin, a first-line drug for type 2 diabetes, remain elusive. Here we report that metformin induces genome-wide alterations in DNA methylation by modulating the activity of S-adenosylhomocysteine hydrolase (SAHH). Exposing cancer cells to metformin leads to hypermethylation of tumor-promoting pathway genes and concomitant inhibition of cell proliferation. Metformin acts by upregulating microRNA let-7 through AMPK activation, leading to degradation of H19 long noncoding RNA, which normally binds to and inactivates SAHH. H19 knockdown activates SAHH, enabling DNA methyltransferase 3B to methylate a subset of genes. This metformin-induced H19 repression and alteration of gene methylation are recapitulated in endometrial cancer tissue samples obtained from patients treated with antidiabetic doses of metformin. Our findings unveil a novel mechanism of action for the drug metformin with implications for the molecular basis of epigenetic dysregulation in cancer. This novel mechanism of action also may be occurring in normal cells.

A naturally occurring secreted human ErbB3 receptor isoform inhibits heregulin-stimulated activation of ErbB2, ErbB3, and ErbB4.

A variety of receptor-mediated signaling pathways are controlled by both positive and negative extracellular regulators. In this study, we demonstrate that a naturally occurring secreted form of the human ErbB3 receptor, p85-soluble ErbB3 (sErbB3), is a potent negative regulator of heregulin (HRG)-stimulated ErbB2, ErbB3, and ErbB4 activation. We show that p85-sErbB3 binds to HRG with an affinity comparable to that of full-length ErbB3 and competitively inhibits high affinity HRG binding to ErbB2/ErbB3 heterodimers on the cell surface of breast carcinoma cells with an IC(50) of 0.5 nM. p85-sErbB3 inhibits HRG-induced phosphorylation of ErbB2, ErbB3, and ErbB4 in breast carcinoma-derived cell lines and can also block HRG-stimulated activation of mitogen-activated protein kinase, Akt, and association of ErbB3 with the phosphatidylinositol 3'-kinase p85 regulatory subunit. Cell growth assays show that exogenous addition of a 100-fold molar excess of p85-sErbB3 inhibits HRG-stimulated cell growth by as much as 90%. Whereas several potential mechanisms of p85-sErbB3 inhibition of ErbB receptor activation exist, our results suggest that at least one means of inhibition is competition for HRG binding. The IC(50) for both p85-sErbB3- and 2C4 (a monoclonal antibody specific for ErbB2)-mediated inhibition of HRG binding is approximately 0.5 nM, although the mechanism of inhibition by these two proteins is distinct. Together these results suggest that p85-sErbB3 is a naturally occurring negative regulator of HRG-stimulated signal transduction that may have important therapeutic applications in human malignancies associated with HRG-mediated cell growth such as breast and prostate cancer.

Isolation and characterization of four alternate c-erbB3 transcripts expressed in ovarian carcinoma-derived cell lines and normal human tissues.

ErbB-3 is a member of the epidermal growth factor receptor (EGFR/ErbB) family. In addition to the previously reported 6.2 kb full-length and 1.4 kb truncated c-erbB3 transcripts, we have observed a 1.7 kb c-erbB3 human transcript in Northern blots that specifically hybridizes to a probe of the extracellular domain of the receptor. Using 3'-RACE we have isolated four novel c-erbB3 cDNA clones of 1.6, 1.7, 2.1 and 2.3 kb from a human ovarian carcinoma-derived cell line. All four alternate transcripts are synthesized by readthrough of an intron and use of an alternative polyadenylation signal within this intron. Identical c-erbB3 transcripts are expressed in normal human placental tissues. Expression of these alternate transcripts is tissue-specific as indicated by Northern blot and RNase protection analyses. Fibroblasts transfected with expression vectors carrying these alternate c-erbB3 cDNA clones stably express truncated ErbB-3 products. Three of these four cDNA clones express a receptor product that is secreted. Immunoprecipitation analysis of primary cultures of human ovarian carcinomas also demonstrate the expression of a 90 kDa ErbB-3 related protein that is secreted. Furthermore, we demonstrate conservation of the exon-intron junctions between members of the erbB gene family in those regions of the gene encoding the extracellular domain. This gene structure is also conserved in the c-erbB1 homologues of Drosophila and C. elegans. Growth regulatory roles for related truncated ErbB products recently have been reported. It is, therefore, possible that the products of these four alternate c-erbB3 transcripts may also play important growth regulatory roles in normal and transformed cells.

Generation of skeletal, smooth and low molecular weight non-muscle tropomyosin isoforms from the chicken tropomyosin 1 gene.

We have determined the organization of the chicken tropomyosin 1 gene by sequencing the cloned genomic DNA. The single-copy gene spans approximately 11,000 bases and includes 12 exons. Comparison of cDNA and genomic sequences demonstrates that three tissue-specific tropomyosins are encoded by the gene: a 284 amino acid skeletal muscle beta-tropomyosin, a 284 amino acid smooth muscle tropomyosin, and a 248 amino acid non-muscle (fibroblast) beta-tropomyosin. Skeletal and smooth muscle transcripts use the same putative promoter and transcription initiation site. However, they are alternatively spliced to generate mRNAs that differ in the region giving rise to amino acids 188 to 213 and 258 through the poly(A) site. The fibroblast transcript uses a promoter, initiation site and first exon that is distinct from that used for both the smooth and the skeletal muscle transcripts. However, beyond the first exon the fibroblast transcript undergoes splicing and polyadenylation that is identical with the smooth muscle transcript.

Sequence, expression and function of an mRNA encoding a soluble form of the human interleukin-6 receptor (sIL-6R).

Soluble receptors have been shown to be potent immunomodulators of their respective ligands. Since IL-6 is a central growth factor for myeloma cells, an sIL-6R may modulate IL-6 activity. We have previously reported a novel IL-6R mRNA from myeloma cells that exhibits a 94-nt deletion of the entire transmembrane domain from codons 356 (G-TG) to 387 (AG-G). The transmembrane domain deletion results in a shift in the translational reading frame with the insertion of 10 new amino acids followed by a stop codon. Sequence analysis shows the ligand-binding domain of the sIL-6R to be identical to that of the membrane-bound IL-6R up to the transmembrane domain deletion. The sIL-6R cDNA was expressed in QT-6 fibroblasts and PA-1 ovarian cells using the expression vector pCDM8. Supernates were immunoprecipitated with anti-IL-6R antibody and cells transfected with the sIL-6R cDNA produced a single band with a molecular weight of 50-55 kDa. This molecular weight corresponds to the size of the sIL-6R protein observed in normal human urine. Supernates were collected from mock or sIL-6R transfected PA-1 cells after 48 hours and assayed for their ability to stimulate or suppress the growth of an IL-6 dependent cell line, ANBL-6. Soluble IL-6R alone had no effect on the growth of the ANBL-6 cells. However, the growth of ANBL-6 cells by sIL-6R was potentiated in the presence of IL-6 and could be blocked by anti-IL-6 antibody. The above results suggest that, in the presence of IL-6, sIL-6R associates with gp130 leading to signal transduction and cell growth.

Reduced expression of the epidermal growth factor signaling system in preeclampsia.

INTRODUCTION: The epidermal growth factor (EGF) signaling system regulates trophoblast differentiation, and its disruption could contribute to perinatal disease. We hypothesized that this pathway is altered in preeclampsia, a disorder associated with trophoblast apoptosis and failure to invade and remodel the uterine spiral arteries. METHODS: Six EGF family peptides and a truncated EGF receptor splice variant (p110/EGFR) were examined using immunohistochemistry in the trophoblast of placentas (N = 76) from women with preeclampsia, and compared to placentas from women of similar gestational age (GA) with preterm labor (PTL) or small for gestational age (SGA) fetuses, as well as normal term placentas. EGF, transforming growth factor-α (TGFA), and heparin-binding EGF-like growth factor (HBEGF) were evaluated using ELISA in maternal plasma from another 20 pregnancies with or without preeclampsia. Cell death was evaluated in the HTR-8/SVneo human cytotrophoblast cell line using TUNEL to evaluate the protective effects of EGF peptides. RESULTS: Trophoblast HBEGF, TGFA, and EGF were significantly reduced in preeclampsia compared to PTL and SGA, while p110/EGFR accumulated significantly on the surface of the chorionic villi (p < 0.05). Plasma EGF levels were significantly decreased in preeclamptic patients, compared to non-preeclamptic patients (p < 0.05). HBEGF, EGF, TGFA, epiregulin, and betacellulin each blocked cytotrophoblast cell death in vitro (p < 0.05). DISCUSSION: Three members of the EGF family are dysregulated in placentas with preeclampsia, whereas p110/EGFR, a potential EGF receptor antagonist, is overexpressed. These findings are consistent with the concept that disruption of the EGF signaling system contributes to aberrant trophoblast development associated with preeclampsia.

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.

The interaction of epidermal growth factor and radiation in human head and neck squamous cell carcinoma cell lines with vastly different radiosensitivities.

PURPOSE: This study was performed to characterize the interaction of epidermal growth factor and radiation in two human head and neck squamous cell cancer cell lines of vastly different radiosensitivities (UM-SCC-6 Radiosensitive; UM-SCC-1 radioresistant). METHODS AND MATERIALS: The two human head and neck squamous cell cancers (UM-SCC-1 and UM-SCC-6) were grown in medium and following the appropriate treatments, cell survival was assessed by a standard colony formation assay. Growth inhibition was assessed by monitoring cell counts following treatment and flow cytometry was used to assess cell cycle distributions. RESULTS AND CONCLUSION: It was determined that exposure to epidermal growth factor (10 ng/ml) for 24 h prior to radiation resulted in radiosensitization in both cell lines, however, the magnitude of radiosensitization was greater in the radiosensitive UM-SCC-6 cells compared to the radioresistant UM-SCC-1 cells. Treatment of the UM-SCC-6 cells with epidermal growth factor (EGF) (10 ng/ml) for 24 h resulted in a growth delay, however, cell growth returned to normal approximately 24 h following removal of EGF. Similar treatment of the UM-SCC-1 cells resulted in no growth inhibition. The 24 h pre-radiation exposures to EGF (10 ng/ml) did not affect the radiation-induced growth delay in either cell line. Additionally, the 24 h exposures to EGF (10 ng/ml) did not affect the radiation-induced growth delay in either cell line. Additionally, the 24 h exposures to EGF (10 ng/ml) did not cause the cells to enter a more radiosensitive cell cycle phase. Further work will be necessary to determine whether events associated with the EGF-induced growth delay in the UM-SCC-6 cells are associated with the enhanced EGF-induced radiosensitization in these cells compared to UM-SCC-1 cells.

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.