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W J Gullick

Publications and source records attributed to W J Gullick.

At least 19 recordsLinked to original sources
PubMed1997

Chemical synthesis, structural modeling, and biological activity of the epidermal growth factor-like domain of human cripto.

Cripto, also known as human teratocarcinoma-derived growth factor 1 (TDGF-1), contains a 40 amino acid region with some similarity to the epidermal growth factor (EGF) domain. However, sequence homology is largely restricted to the classical cysteine/glycine motif with only limited similarities in other regions. Significant differences to human EGF include the absence of all seven residues between the two N-terminal half-cystines and a five-residue shorter loop between the third and fourth half-cystines. We examine the hypothesis that, in spite of these differences, cripto can adopt the characteristic EGF-like 1-3, 2-4, 5-6 disulfide bond pattern. A comparative structural model of the growth factor cripto was constructed on the basis of its similarity to EGF, transforming growth factor alpha (TGF-alpha), and the EGF-like domain of human clotting factor IX. The predicted disulfide bridges and disulfide-bridged loops were analyzed and appear viable in the modeled structure. Moreover, to ascertain the importance of disulfide arrangement for cripto bioactivity, two 47-residue peptides were synthesized and then refolded using either a simple oxidative or a controlled sequential refolding protocol. The cripto peptides were tested for their ability to stimulate MAP-kinase activity, for inhibition of beta-casein induction, and for Shc phosphorylation in MDA-MB 453 human mammary carcinoma cells and HC-11 mouse mammary epithelial cells. Data suggest that cripto does adopt the 1-3, 2-4, 5-6 disulfide pattern and thus forms the classical EGF-like fold in spite of the significant deletions within the folding domain. The predicted structure of cripto shows some of the characteristics of both the ErbB1- and ErbB3/ErbB4-binding growth factors.

Amino Acid Sequence↗
PubMed1997

Cripto enhances the tyrosine phosphorylation of Shc and activates mitogen-activated protein kinase (MAPK) in mammary epithelial cells.

Cripto-1 (CR-1), a recently discovered protein of the epidermal growth factor (EGF) family, was found to interact with a high affinity, saturable binding site(s) on HC-11 mouse mammary epithelial cells and on several different human breast cancer cell lines. This receptor exhibits specificity for CR-1, since other EGF-related peptides including EGF, transforming growth factor alpha, heparin-binding EGF-like growth factor, amphiregulin, epiregulin, betacellulin, or heregulin beta1 that bind to either the EGF receptor or to other type 1 receptor tyrosine kinases such as erb B-3 or erb B-4 fail to compete for binding. Conversely, CR-1 was found not to directly bind to or to activate the tyrosine kinases associated with the EGFR, erb B-2, erb B-3, or erb B-4 either alone or in various pairwise combinations which have been ectopically expressed in Ba/F3 mouse pro-B lymphocyte cells. However, exogenous CR-1 could induce an increase in the tyrosine phosphorylation of 185- and 120-kDa proteins and a rapid (within 3-5 min) increase in the tyrosine phosphorylation of the SH2-containing adaptor proteins p66, p52, and p46 Shc in mouse mammary HC-11 epithelial cells and in human MDA-MB-453 and SKBr-3 breast cancer cells. CR-1 was also found to promote an increase in the association of the adaptor Grb2-guanine nucleotide exchange factor-mouse son of sevenless (mSOS) signaling complex with tyrosine-phosphorylated Shc in HC-11 cells. Finally, CR-1 was able to increase p42(erk-2) mitogen-activated protein kinase (MAPK) activity in HC-11 cells within 5-10 min of treatment. These data demonstrate that CR-1 can function through a receptor which activates intracellular components in the ras/raf/MEK/MAPK pathway.

Animals↗
PubMed1997

Expression of epidermal growth factor-related proteins in the aged adult mouse mammary gland and their relationship to tumorigenesis.

Mammary glands from female BALB/c mice of different ages and parity were screened for production of three epidermal growth factor (EGF) related transforming growth factors and their corresponding mRNAs. Glands were obtained from 2-26-month-old nulliparous, 4-26-month-old parous, and 2-8-month-old midpregnant mice. Reverse-transcribed polymerase chain reaction (RT-PCR) was used to screen for mRNA from the transforming growth factor alpha (TGF alpha), cripto-1 (CR-1), and amphiregulin (AR) genes in extracts of whole mammary glands. TGF alpha, CR-1, and AR transcripts were detected in all of the mammary glands assayed. In situ hybridization was then used to localize these mRNAs among various cell types in sections of glands. TGF alpha mRNA levels were low in the mammary epithelium from young nulliparous mice, high in the stroma of midpregnant mammary glands, and highest in luminal epithelium of the aged glands. AR mRNA levels were high and remained unchanged in all developmental stages. CR-1 mRNA level increased with age and was detected primarily in epithelium, with some scattered expression in adjacent stroma. Finally, TGF alpha, CR-1, and AR proteins were immunolocalized in histological sections of mammary glands from the various developmental stages. TGF alpha was detected sporadically in midpregnant mice, with more conspicuous reactivity seen in 18-26-month-old mice (38% of mice). CR-1 immunoreactivity was detected in 100% of the 18-26-month-old glands but not in any other age groups. Strong AR immunoreactivity was observed in in all glands, including 100% of the 18-26-month-old glands. Staining for all three of these growth factors was observed primarily in the epithelium, with some reactivity detected in the periductal fibroblasts. No significant difference was discerned between glands from nulliparous and parous animals. We also found intense CR-1 and AR mRNA expression and strong immunoreactivity in seven different carcinogen-induced and eight spontaneous mammary tumors. Our results demonstrate that these growth factors accumulate in significant amounts in the old gland of both nulliparous and parous mice. The observations suggest that these growth factors are positioned to contribute to abnormal development in the older mammary gland, predisposing them to tumorigenesis.

Aging↗
PubMed1997

A potential autocrine loop between heregulin-alpha and erbB-3 receptor in human prostatic adenocarcinoma.

OBJECTIVE: To examine the expression of heregulin-alpha (a new member of the epidermal growth factor family) and its receptor erbB-3 in human prostate cancer by immunohistochemistry, and to analyse their implication for clinical outcome. PATIENTS AND METHODS: Using specific anti-peptide antibodies, tumour samples from 50 consecutive patients with newly diagnosed prostate cancer (18 well, 15 moderately and 17 poorly differentiated) and four patients with benign prostatic hyperplasia (BPH) were immunostained for heregulin-alpha (an isoform of heregulin) and erbB-3 proteins. Representative areas from each case were used to assess immunoreactivity. RESULTS: Heregulin-alpha was expressed (i.e. > or = 10% of tumour cells positive) in 36 (72%) and erbB-3 in 27 (54%) of the 50 cases. High levels of expression of heregulin-alpha (> 90% of tumour cells stained) appeared to be related to high-grade tumours, but the association did not reach statistical significance. There was no correlation between the levels of expression of erbB-3 and tumour grade. All four samples from BPH stained negatively for heregulin-alpha and erbB-3. Fourteen patients had bony metastases identified by bone scintigraphy and all received androgen ablation after undergoing transurethral resection of the prostate. Immunoreactivity for heregulin-alpha and erbB-3 were absent in five cases, all of whom responded to hormone manipulation and remained symptom-free at the time of review (mean follow up 3 years, range 2-4). Despite hormone manipulation, the nine patients overexpressing heregulin-alpha and/or erbB-3 died during a mean follow up of 2.5 years (range 0.5-4). CONCLUSION: The overexpression of heregulin-alpha and erbB-3 in prostate cancer is detectable by immunostaining. Activation of the type 1 growth-factor receptor system by heregulin-alpha and/or the erbB-3 receptor appears to be associated with a less favourable prognosis in advanced disease.

Adenocarcinoma↗
PubMed1996

Intracellular expression of a single-chain antibody directed to the EGFR leads to growth inhibition of tumor cells.

A gene encoding a single-chain antibody (scFv) which specifically binds the epidermal growth factor receptor (EGFR) has been constructed from hybridoma cells producing the R1 monoclonal antibody. The gene, designated scFv-R1R, was introduced into EGFR transformed NIH3T3 cells via retroviral infection. scFv-R1R was directed to the lumen of the endoplasmic reticulum (ER) where it bound the extracellular domain of the receptor inhibiting its appearance on the plasma membrane. In these cells, EGF induced tyrosine phosphorylation of the EGFR and several substrates was greatly reduced. Furthermore, intracellular retention of EGFR caused a partial inhibition in the transformed growth of the cells. Intracellular expression of receptor tyrosine kinase directed scFvs is a novel approach for affecting tumor cell growth. We have recently shown that scFv-5R directed to ErbB2, another member of the ErbB family, blocks the anchorage independent growth of ErbB2 transformed cells. In order to examine the effects of scFv-R1R and scFv-5R on the long-term growth of tumor cells overexpressing either EGFR or ErbB2, retroviruses encoding the two scFvs were used to infect various human tumor cell lines. Intracellular expression of the scFvs resulted in a marked inhibition of stable colony formation in some of the cell lines. In general, inhibition was observed when overexpressed receptor was targeted. However, in some cases expression of both scFvs was incompatible with long term cell growth suggesting that heterodimers of ErbB2 and EGFR are essential for the growth of some human tumor cell lines.

3T3 Cells↗
PubMed1996

Expression of the type 1 tyrosine kinase growth factor receptors EGF receptor, c-erbB2 and c-erbB3 in bladder cancer.

Expression of the c-erbB3 protein was determined in transitional cell carcinoma of the bladder by immunohistochemistry. Strong membrane staining was observed in 10 per cent of cases (7/70) and cytoplasmic and membrane overexpression in 20 per cent (14/70). Overexpression of the epidermal growth factor (EGF) receptor (36 per cent, 25/70) and c-erbB2 proteins (9 per cent 6/70) was determined in the same series of cases. c-erbB3 overexpression was positively correlated with EGF receptor expression (P < 0.025) but appeared to be inversely associated with c-erbB2 overexpression.

Biomarkers, Tumor↗
PubMed1996

C-erbB-3 in human breast carcinoma: expression and relation to prognosis and established prognostic indicators.

A series of 346 patients with primary operable breast cancer and a series of 145 patients with advanced breast cancer were investigated for c-erbB-3 protein expression using the monoclonal antibody RTJ1. Formalin-fixed, paraffin-embedded tumour samples were stained using a standard immunochemical method and staining was assessed on a four-point scale. The study aimed to observe the expression of the c-erbB-3 protein and investigate any relationship between expression and established prognostic indicators and prognosis. In both the primary and advanced series breast tumour tissue was found to stain heterogeneously for c-erbB-3. The staining was observed to be predominantly cytoplasmic and the majority of tumours exhibited moderate positivity. However, 15% and 35% of cases in the primary operable and advanced series respectively displayed strong positive staining. No significant difference was found between the staining in the primary and advanced series. In the primary operable breast cancers, no significant associations were demonstrated with overall survival, disease-free interval, regional recurrence, the presence of distant metastases, age, menopausal status, oestrogen receptor status, histological grade, lymph node stage, vascular invasion and c-erbB-2 protein expression. However, a significant association was seen between the degree of c-erbB-3 immunoreactivity and both tumour size (P < 0.01) and tumour type prognostic group (P = 0.05). No overall association with local recurrence was seen when the four groups of c-erbB-3 expression were analysed (P = 0.12), but when those tumours showing no or weak staining were compared with those showing moderate and strong immunoreactivity it was seen that the latter were significantly more likely to develop local recurrence (P = 0.03). In the series of patients with advanced disease, no significant associations were demonstrated with survival, UICC criteria, age, menopausal status, oestrogen receptor status, histological grade, c-erbB-2 status or the presence of vascular invasion. In conclusion this study found variable expression of c-erbB-3 protein in human breast carcinoma and an association with some recognised prognostic factors in those patients with primary operable breast carcinoma. It seems, however, unlikely that c-erbB-3 protein expression will emerge as a powerful enough prognostic factor to be of value in clinical practice.

Adult↗
PubMed1996

The c-erbB3/HER3 receptor in human cancer.

c-erbB3 is a receptor for NDF/heregulin that can engage in heterodimerization with other type I receptor tyrosine kinases. Despite very limited kinase activity, it can stimulate responses in heterodimers not evoked by other family members. It is overexpressed frequently in several common solid tumours and thus represents a target for new forms of therapies.

Animals↗
PubMed1995

Specific targeting of a mutant, activated FGF receptor found in glioblastoma using a monoclonal antibody.

A truncated epidermal growth factor receptor (EGFR) expressed from a rearranged and amplified EGFR gene is present at high frequency in gliomas. In this work we show that when this receptor is expressed in NIH3T3 fibroblasts it is partially activated and confers tumorigenicity to this cell line in vivo but no growth advantage in in vitro anchorage-independent growth assays. Because the mutation occurs in the extracellular domain of the receptor, it can be considered to represent a glioma-specific tumour marker. Here we demonstrate that 2 monoclonal antibodies, DH1.1 and DH8.3, raised to a synthetic peptide spanning the unique junctional sequence, can recognise the mutant receptor but not the normal receptor in both denatured and native states. Furthermore, radiolabelled antibody DH8.3 successfully targets tumours expressing this antigen in nude mice.

3T3 Cells↗
PubMed1995

Binding of Shc to the NPXY motif is mediated by its N-terminal domain.

Shc is an SH2-containing adapter protein that binds to and is phosphorylated by a large number of growth factor receptors. Phosphorylated Shc is able to interact with the Grb2-Sos complex which is responsible for mediating nucleotide exchange on Ras. We have shown previously that binding of Shc to the epidermal growth factor (EGF)-like receptor, c-ErbB-3, is through an NPXY motif (Prigent, S. A., and Gullick, W. J. (1994) EMBO J. 13, 2831-2841) shared by middle T antigen, TrkA, and EGF receptor. It has recently been reported that a region distinct from the SH2 domain is able to bind to tyrosine-phosphorylated proteins. In this paper we have used fusion proteins of various Shc domains to show that it is the N-terminal domain of Shc that is primarily responsible for binding EGF receptor and c-ErbB-3. Furthermore, by competition studies with synthetic phosphopeptides we have shown that this N-terminal domain binds to the previously identified NPXY motif.

3T3 Cells↗
PubMed1995

Detection and location of amphiregulin and Cripto-1 expression in the developing postnatal mouse mammary gland.

Amphiregulin (Ar) and Cripto-1 (Cr-1) are growth promoting peptides that share amino acid sequence homology with epidermal growth factor (EGF). The present study examined Ar and Cr-1 mRNA and protein expression during various stages of C57BL/6 mouse mammary morphogenesis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect transcripts for Ar and Cr-1 at all stages of mammary development. Immunocytochemical (ICC) localization demonstrated that in virgin 4-week to mature 12-week-old mouse fourth inguinal mammary gland, Ar and Cr-1 are expressed in the stromal cells, luminal epithelial cells, and myoepithelial cells of the branching ducts. Ar, and to lesser extent Cr-1, were also found in the epithelial cap cells and in the luminal epithelial cells of the advancing terminal end bud (TEB) from virgin 4-week and 6-week-old mice. Western blot analysis demonstrated that both Ar (28 and 26 kDa) and Cr-1 (90, 67, 56, and 21 kDa) proteins are expressed in virgin, 13.5 day midpregnant and in the 14 day lactating mammary gland. In addition, Ar and Cr-1 are associated with developing alveolar structures as determined by ICC. These results imply that together with EGF and transforming growth factor alpha (TGF alpha), Ar and Cr-1 may play salient roles as modifiers in the morphogenesis and differentiation of the mammary gland.

Amphiregulin↗
PubMed1995

Association of epidermal growth factor-related peptides and type I receptor tyrosine kinase receptors with prognosis of human colorectal carcinomas.

The frequency of expression and localization of cripto-1 (CR-1), amphiregulin (AR), transforming growth factor alpha (TGF alpha), epidermal growth factor receptor (EGFR) and erbB-2 were examined by immunohistochemistry in 45 carcinomas and adjacent non-involved normal colon mucosa. Thirty (66.7%), 24 (53.3%), 23 (51.1%), 23 (51.1%) and 13 (28.9%) of the 45 carcinomas showed positive staining for CR-1, AR, TGF alpha, EGFR and erbB-2, respectively, whereas 7 (15.5%), 17 (37.7%), 15 (33.3%), 20 (44.4%) and 0 (0%) of the corresponding non-involved normal mucosa specimens were reactive. Among 13 carcinomas with lymph node involvement, 10 (76.9%), 8 (61.5%), 10 (76.9%), 8 (61.5%) and 7 (53.8%) exhibited positive staining for CR-1, AR, TGF-alpha, EGFR and erbB-2, respectively. There was a statistically significant association between the frequency of either TGF alpha (P < 0.05) or erbB-2 (P < 0.05) expression and lymph node metastasis. In addition, a significantly higher frequency of positive staining for TGF alpha was observed in Dukes' grade C carcinomas (P < 0.05). Finally, significant trends for coexpression of EGFR and either TGF alpha (P < 0.01) or AR (P < 0.05) were detected in carcinomas. These data suggest that AR and TGF alpha may play an important role in the development of colorectal carcinomas through an autocrine mechanism involving EGFR, and demonstrate that TGF alpha and erbB-2 may be more reliable indicators of metastasis or prognosis than CR-1, AR or EGFR in human colon cancers.

Adenocarcinoma↗
PubMed1994

Expression and functional activity of fibroblast growth factors and their receptors in human pancreatic cancer.

We have analysed expression of the first 7 members of the family of heparin-binding fibroblast growth factor (FGFs) and their 4 high-affinity receptors (FGFRs) in human pancreatic carcinoma cell lines, both at the mRNA and protein levels. In cell lines expressing FGFRs, 2 typical patterns were observed: (i) expression of FGFR-I, -3 or -4 along with the expression of at least one FGF; (ii) co-expression of FGFR-3 and FGFR-4 in the absence of FGF expression. Using RT-PCR, transcripts representing multiple isoforms of both extracellular and intracellular domains of FGFR-I were detected in the cell line PT45. A novel extracellular domain variant of FGFR-I was predicted to encode the first immunoglobulin loop in a potentially secreted form. Protein expression of the splice variants of FGFR-I was confirmed by immunoprecipitation with specific antibodies in radiolabelled ligand cross-linking experiments. The type I carboxyl end and the alpha subtype extracellular domain were detected in the PANC-I cell line, while the type I carboxyl terminus and the gamma subtype extracellular domain were expressed in the PT45 cell line. Expression of FGF-2 in PT45 was also detected by immunoprecipitation using 3 different anti-FGF-2 antibodies. Apart from the 18-kDa product, higher molecular weight isoforms, namely 22- and 23-kDa isoforms, were expressed. In an assay of anchorage-independent growth, exogenous FGF-2 stimulated a maximum 15-fold and 10-fold increase in colony formation by the cell lines MIA PACA-2 and PANC-I respectively. Treatment of monolayer cultures of the same cell lines did not promote growth. However, a specific neutralising antibody against FGF-2 reduced cell proliferation of MIA PACA-2 cells by 50%.

Base Sequence↗
PubMed1994

NDF/heregulin stimulates the phosphorylation of Her3/erbB3.

Her3/erbB3 has been identified as a third member of the epidermal growth factor receptor (EGFR) family [(1989) Proc. Natl. Acad. Sci. USA 86, 9193-9197; (1990) Proc. Natl. Acad. Sci. USA 87, 4905-4909]. The natural ligand for Her3 has not been identified. Although recently NDF has been proposed as a specific ligand for Her4 [(1993) Nature 366, 473-475; (1993) J. Biol. Chem. 268, 18407-18410], we report here that Her3 was phosphorylated on tyrosine not only in three breast carcinoma cell lines, MDAMB453, MDAMB468 and SKBR3, but also in Her3-transfected CHO cells in response to NDF stimulation. In further studies, cells were reacted with 125I-labeled NDF and then chemically crosslinked. Immunoprecipitation with anti-Her3 revealed a dense high Mw band, greater than 400 kDa. The results suggest that NDF may be a ligand of Her3 and induces receptor hetero-oligomerization.

Animals↗
PubMed1994

Identification and biological characterization of an epidermal growth factor-related protein: cripto-1.

The human and mouse cripto-1 (CR-1) genes can code for proteins related in structure to epidermal growth factor (EGF). A specific 36-kDa immunoreactive protein was detected by Western blot analysis in human cell lines that express CR-1 mRNA but not in cell lines that fail to express this transcript. Immunoprecipitation of GEO colon carcinoma or mouse embryonal carcinoma cells detected 27-29-kDa and 24-kDa proteins, respectively. Cell lysates and conditioned medium that were prepared from several CHO clones and were expressing either a recombinant human or mouse CR-1 cDNA contained immunospecific 27-29-kDa and 24-kDa proteins, respectively. Monensin or tunicamycin treatment resulted in a shift of the 27-29-kDa human CR-1 protein to 24 kDa and 20 kDa, respectively. The 20-kDa protein was also observed after digestion of the 27-29-kDa human CR-1 protein with N-glycosidase F. Using two CR-1 synthetic refolded peptides that correspond to the EGF-like domain of the human CR-1 sequence or conditioned medium obtained from human CR-1 expressing CHO cells, growth stimulatory activity could be detected on non-transformed human mammary epithelial cells and on two human breast cancer cell lines. EGF receptor-blocking antibody did not inhibit the growth stimulatory action of the CR-1 protein. Likewise, the CR-1 refolded peptides or conditioned medium from the human CR-1-expressing CHO cells failed to inhibit the binding of 125I-EGF in an EGF-radioreceptor assay. These data demonstrate that the CR-1 is a glycoprotein that can function as a growth factor through an EGF receptor-independent pathway.

Amino Acid Sequence↗
PubMed1994

Identification of c-erbB-3 binding sites for phosphatidylinositol 3'-kinase and SHC using an EGF receptor/c-erbB-3 chimera.

c-erbB-3 is a member of the type I (EGF receptor-related) family of growth factor receptors for which no ligand has been identified. To facilitate ligand stimulation we have constructed a chimeric receptor which possesses an activatable kinase and promotes the growth of NIH 3T3 fibroblasts. In this study we have shown that SHC and phosphatidylinositol 3'-kinase bind to the activated EGF receptor/c-erbB-3 chimera. Whereas p85 is not phosphorylated to a significant extent, SHC appears to be a major substrate for phosphorylation on tyrosine. In contrast to EGF receptor and c-erbB-2, we were unable to detect binding of activated c-erbB-3 to GRB2. Using synthetic peptides corresponding to each of 13 potential phosphorylation sites on c-erbB-3, we have shown that tyrosine 1309 is responsible for SHC binding. Peptides containing the motif YXXM inhibit p85 association. By comparison with recently reported SHC binding sites on Middle T antigen and Trk we have identified a SHC binding motif, NPXY.

Adaptor Proteins, Signal Transducing↗
PubMed1994

The type I growth factor receptors in human breast cancer.

The type 1 family of growth factor receptors includes the EGFR, c-erbB2, c-erbB3, and c-erbB4. All four members of the family are expressed in breast cancer. The EGFR gene and more frequently the c-erbB2 gene are amplified in a proportion of cases. In addition to increased expression as a result of gene amplification, overexpression of perhaps all of the receptors also appears to occur, probably as a result of increased mRNA transcription. Overexpression may have prognostic value and may predict response to current therapies. Finally these GFR proteins represent targets for new types of chemotherapeutic agents.

Animals↗