Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-alpha, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-alpha mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-alpha protein by DLD-1 and CoLo320DM cells was confirmed by TGF-alpha specific monoclonal antibody binding assay. The spontaneous 3H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-alpha monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-alpha produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.     

Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-α

Tumor necrosis factor (TNF)-α has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-α. Among the cell lines tested those resistant to TNF-α were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin β1 resulted in a significant increase in the number of cells surviving TNF-α treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-α. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-α retained TNF-α activity. This scFv(14E1)-TNF-α molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-κB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays. Received: 6 May 1998 / Accepted: 16 July 1998     

The enlargement of the hormone immune deprivation concept to the blocking of TGFα-autocrine loop: EGFR signaling inhibition

Transforming growth factor alpha (TGFα) is a potent ligand of the epidermal growth factor receptor (EGFR). EGFR is frequently over-expressed in epithelial tumors and endogenous ligands, mostly TGFα, are frequently co-expressed with EGFR, potentially resulting in autocrine stimulation of tumor cell growth. Therefore, different therapeutic approaches aim for the inactivation of TGFα/EGF/EGFR signaling system, but no approach is based on TGFα as a target. The principal goal of this work was to assess the potential of an active specific immunotherapy approach to block the TGFα/EGFR autocrine loop. For the proof of the concept, a fusion protein between human TGFα (hTGFα) and P64k protein from Neisseria meningitidis was generated, and its immunogenicity characterized in a mouse model using different adjuvants. All immunogens were effective for the generation of specific humoral responses against hTGFα. The inmunodominant epitope of hTGFα when immunizing mice with the fusion protein involved the C-loop/C-terminal region. This region includes key residues for hTGFα binding to EGFR. The anti-hTGFα immune mice sera recognized the natural hTGFα precursor in A431 cells and hTGFα-transfected 3T3 fibroblasts as revealed by flow cytometry analysis and immunoblotting. They inhibited the binding of ¹²⁵I-TGFα to the EGFR, EGFR-autophosphorylation, and downstream activation of MAP kinases as well as proliferation of two EGFR-expressing human carcinoma cell lines. These data suggest that EGFR signaling activation by the hTGFα autocrine loop may be inhibited in vivo by induction of specifically blocking antibodies. The fusion protein reported in this paper could be a potential immunogen for the development of a new cancer vaccine. Part of this work was supported by a travel scholarship sponsored by the Boehringer Ingelheim Fonds     

Mutual regulation of TGF-β1, TβRII and ErbB receptors expression in human thyroid carcinomas

The role of EGF and TGF-β1 in thyroid cancer is still not clearly defined. TGF-β1 inhibited the cellular growth and migration of follicular (FTC-133) and papillary (B-CPAP) thyroid carcinoma cell lines. Co-treatments of TGF-β1 and EGF inhibited proliferation in both cell lines, but displayed opposite effect on their migratory capability, leading to inhibition in B-CPAP and promotion in FTC-133 cells, by a MAPK-dependent mechanism. TGF-β1, TβRII and EGFR expressions were evaluated in benign and malignant thyroid tumors. Both positivity (51.7% and 60.0% and 80.0% in FA and PTC and FTC) and overexpression (60.0%, 77.7% and 75.0% in FA, PTC and FTC) of EGFR mRNA correlates with the aggressive tumor behavior. The moderate overexpression of TGF-β1 and TβRII mRNA in PTC tissues (61.5% and 62.5%, respectively), counteracted their high overexpression in FTC tissues (100% and 100%, respectively), while EGFR overexpression was similar in both carcinomas. Papillary carcinomas were positive to E-cadherin expression, while the follicular carcinomas lose E-cadherin staining. Our findings of TGF-β1/TβRII and EGFR overexpressions together with a loss of E-cadherin observed in human follicular thyroid carcinomas, and of increased migration ability MAPK-dependent after EGF/TGF-β1 treatments in the follicular thyroid carcinoma cell line, reinforced the hypothesis of a cross-talk between EGF and TGF-β1 systems in follicular thyroid carcinomas phenotype.     

Growth factor interactions between mouse mammary cell lines cocultured in collagen gels

Summary Three related mouse mammary cell lines were cultured in collagen gels and assayed for growth factor responsiveness and interaction via soluble factors. The CL-S1 cell line is nontumorigenic and grows poorly in collagen gel culture. The +SA and −SA cell lines exhibit different degrees of malignant behavior in vivo and have different growth properties in vitro. In collagen gel culture, +SA growth was stimulated by serum but not by epidermal growth factor (EGF), whereas both serum and EGF were required for optimal growth of −SA cells of early passage number as well as CL-S1 cells. −SA cells of later passage repeatedly exhibited a change so as to no longer require serum while retaining EGF responsiveness. [¹²⁵I]EGF binding analyses indicated that CL-S1 cells bound EGF with less affinity than did −SA cells whereas +SA cells bound almost to ligand. When cell lines were maintained in separate collagen gels but shared the same culture medium, growth of +SA or −SA cells was slightly enhanced in the presence of CL-S1 cells and −SA cell growth was enhanced by the presence of +SA cells. Using the normal rat kidney fibroblast line NRK (clone 49F) as an indicator, serum-containing conditioned media from each cell line and from each pair of cell lines cultured in collagen gels were tested for transforming growth factor (TGF) activity. Both the −SA and CL-S1 lines tested positive for TGF-α production and possibly released a TGF-β activity. These results suggest mechanisms by which cell populations in and around tumors can modify one another’s growth characteristics. The work was supported by a grant from the American Institute for Cancer Research, by American Cancer Society Institutional grant IN-119, by funds from the Poncin Trust (Seattle-First National Bank), and by grants CA-39611 and CA46885 from the National Institutes of Health, Bethesda, MD.     

Expression of intermediate filaments,EGF and TGF-α in early human kidney development

The spatial and temporal expression patterns of cytokeratins, vimentin, epithelial growth factor (EGF) and transforming growth factor alpha (TGF-α), were investigated in the 5–9-week old human mesonephros and metanephros. Vimentin was found in all mesonephric structures, while cytokeratins were seen only in the mesonephric tubules. EGF and TGF-α were detected early in all mesonephric structures, and immunoreactivity to both factors decreased in later stages. In the 5–6-week metanephros, vimentin immunoreactivity was found in all structures and later increased in the collecting system and interstitium. In the 5th week, cytokeratins 8 and 19 appeared in the ureteric bud and ampullae, and later showed increasing immunoreactivity in the collecting system and nephrons. The coexpression of intermediate filament proteins in metanephric development is a temporary feature and might be associated with mesenchymal to epithelial transformation of developing nephrons. In adult kidneys, such coexpression is associated with fibrosis or carcinomatous changes. At early stages, immunoreactivity to EGF and TGF-α was detected in all metanephric structures and from the 7th week onward, it decreased in differentiating nephrons. EGF and TGF-α patterns of appearance indicate their role in induction, proliferation and growth of metanephric structures. Disturbances in that pattern might cause reduction in kidney growth.     

TGF-alpha as a candidate tumor antigen for renal cell carcinomas

Objectives  Patients with renal cell carcinomas (RCC) have few treatment options, underscoring the importance of developing new approaches such as immunotherapy. However, few tumor associated antigens (TAA), which can be targeted by immunotherapy, have been identified for this type of cancer. von Hippel-Lindau clear cell RCC (VHL^−/−RCC) are characterized by mutations in the VHL tumor suppressor gene. Loss of VHL function causes the overexpression of transforming growth factor (TGF)-α, leading us to hypothesize that TGF-α could be a potential TAA for immunotherapy of kidney cancer, which was evaluated in this study. Methods and results  We first confirmed the absent or weak expression of TGF-α in important normal tissues as well as its overexpression in 61% of renal tumors in comparison to autologous normal kidney tissues. In addition, we demonstrated the immunogenicity of TGF-α, by expanding many T cell lines specific for certain TGF-α peptides or the mature TGF-α protein, when presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. Interestingly, some of these TGF-α-specific T cells were polyfunctionals and secreted IFN-γ, TNF-α and IL-2. Conclusion  We have shown that TGF-α is a valid candidate TAA, which should allow the development of a targeted immunotherapy.     

Expression of Epidermal Growth Factor Receptor Detected by Cetuximab Indicates Its Efficacy to Inhibit In Vitro and In Vivo Proliferation of Colorectal Cancer Cells

Cetuximab is a chimeric mouse–human monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). However, EGFR expression determined by immunohistochemistry does not predict clinical outcomes of colorectal cancer (CRC) patients treated with cetuximab. Therefore, we evaluated the correlation between EGFR levels detected by cetuximab and drug sensitivities of CRC cell lines (Caco-2, WiDR, SW480, and HCT116) and the A431 epidermoid carcinoma cell line. We used flow cytometry (FCM) to detect EGFR-binding of biotinylated cetuximab on the cell surface. Subcloned cell lines showing the highest and lowest EGFR expression levels were chosen for further study. Cytotoxic assays were used to determine differential responses to cetuximab. Xenograft models treated with cetuximab intraperitoneally to assess sensitivity to cetuximab. Strong responses to cetuximab were specifically exhibited by subcloned cells with high EGFR expression levels. Furthermore, cetuximab inhibited the growth of tumors in xenograft models with high or low EGFR expression levels by 35% and 10%–20%, respectively. We conclude that detection of EGFR expression by cetuximab promises to provide a novel, sensitive, and specific method for predicting the sensitivity of CRC to cetuximab.     

EGFR expression and EGF stimulation of proliferation in human liver carcinoma cells

Epidermal growth factor receptor (EGFR) gene expression and growth stimulation of EGF on human hepatoma cells of cell lines BEL-7404 and SMMC-7721 were studied. 125I-EGF binding assay was used to measure the binding characteristics and the amounts of EGFR on these cells. The binding time course and the binding competition assay showed that the binding of 125I-EGF to 7404 cells was saturable and specific. Scatchard analysis of EGF binding curve indicated that 7404 and 7721 cells expressed approximately 1.1 x 10(5) and 0.7 x 10(5) EGFRs per cell with binding affinity (Kd) 2.1 nM and 1.8 nM respectively. Northern hybridization and immunoblotting analysis showed the EGFR gene expression products in 7404 and 7721 cells were 5.6 Kb mRNA and 170 Kilo-dalton glycoprotein. Anchorage-dependent growth of 7404 and 7721 cells was stimulated in the presence of nanogram quantities of EGF in medium containing 10% calf serum or 0.5% calf serum. The factors in serum appeared to act synergitically in stimulating of cell proliferation. EGF also stimulated the anchorage-independent growth of 7404 and 7721 cells in soft agar. The results suggest that EGFR is actively expressed in human hepatoma 7404 and 7721 cells and EGF may be one of the mitogens needed for the growth of hepatoma cells.     

Characteristics of EGFR family-mediated HRG signals in human ovarian cancer

The ability of the epidermal growth factor receptor (EGFR) family members, EGFR, HER2, HER3, and HER4, to form homo- and heterodimers after interaction with different ligands expands the signal diversity of these proteins. We investigated their mechanism of activation by exogenous EGF and heregulin (HRG) in human ovarian carcinoma cell lines which express different amounts and combinations of the four receptors. Consistently the predominant interaction after EGF treatment was between EGFR and HER2, whereas activation of HER3 and HER4 depended on the relative abundance of the four receptors in the cells. Remarkably HER3 activation by HRG could occurs independent of HER2, and in one cell line almost no HER4 activation by HRG was detected despite high levels expression. Both EGF and HRG induced activation of mitogen-activated protein kinase (MAPK), but the time course of MAPK activation differed depending on the hetero-dimers induced. EGF and HRG mediated cell growth through the EGFR/HER2 heterodimer and HER4, respectively, but not through HER3 when it was the only HRG receptor expressed and phosphorylated in the cells. These findings reveal a distinct pattern of HRG induced EGFR family interaction in ovarian cancer that is distinct from that described in human breast cancer. Moreover EGF and HRG can exert distinct biological functions depending on the receptor complexes induced in a given ovarian cancer cell line.     

An Inducible TGF-β2-TGFβR Pathway Modulates the Sensitivity of HNSCC Cells to Tyrosine Kinase Inhibitors Targeting Dominant Receptor Tyrosine Kinases

The epidermal growth factor receptor (EGFR) is overexpressed in approximately 90% of head and neck squamous cell carcinomas (HNSCC), and molecularly targeted therapy against the EGFR with the monoclonal antibody cetuximab modestly increases overall survival in head and neck cancer patients. We hypothesize that co-signaling through additional pathways limits the efficacy of cetuximab and EGFR-specific tyrosine kinase inhibitors (TKIs) in the clinical treatment of HNSCC. Analysis of gene expression changes in HNSCC cell lines treated 4 days with TKIs targeting EGFR and/or fibroblast growth factor receptors (FGFRs) identified transforming growth factor beta 2 (TGF-β2) induction in the three cell lines tested. Measurement of TGF-β2 mRNA validated this observation and extended it to additional cell lines. Moreover, TGF-β2 mRNA was increased in primary patient HNSCC xenografts treated for 4 weeks with cetuximab, demonstrating in vivo relevance of these findings. Functional genomics analyses with shRNA libraries identified TGF-β2 and TGF-β receptors (TGFβRs) as synthetic lethal genes in the context of TKI treatment. Further, direct RNAi-mediated silencing of TGF-β2 inhibited cell growth, both alone and in combination with TKIs. Also, a pharmacological TGFβRI inhibitor similarly inhibited basal growth and enhanced TKI efficacy. In summary, the studies support a TGF-β2-TGFβR pathway as a TKI-inducible growth pathway in HNSCC that limits efficacy of EGFR-specific inhibitors.     

NADPH Oxidase NOX1 Controls Autocrine Growth of Liver Tumor Cells through Up-regulation of the Epidermal Growth Factor Receptor Pathway

FaO rat hepatoma cells proliferate in the absence of serum through a mechanism that requires activation of the epidermal growth factor receptor (EGFR) pathway. The aim of this work was to analyze the molecular mechanisms that control EGFR activation in these and other liver tumor cells. Reactive oxygen species production is observed a short time after serum withdrawal in FaO cells, coincident with up-regulation of the NADPH oxidase NOX1. NOX1-targeted knockdown, the use of antioxidants, or pharmacological inhibition of NADPH oxidase attenuates autocrine growth, coincident with lower mRNA levels of EGFR and its ligand transforming growth factor-α (TGF-α) and a decrease in phosphorylation of EGFR. EGFR-targeted knockdown induces similar effects on cell growth and downstream signals to those observed in NOX1-depleted cells. Early NOX1 activation induces both a feedback-positive loop via an Src-ERK pathway that up-regulates its own levels, and a parallel signaling pathway through p38 MAPK and AKT resulting in EGFR and TGF-α up-regulation. Human hepatocellular carcinoma cell lines, but not non-tumoral hepatocytes, show autocrine growth upon serum withdrawal, which is also coincident with NOX1 up-regulation that mediates EGFR and TGF-α expression. The use of antioxidants, or pharmacological inhibition of NADPH oxidase, effectively attenuates autocrine growth in hepatocellular carcinoma cell lines. In summary, results presented in this study indicate that NOX1 might control autocrine cell growth of liver tumor cells through regulation of the EGFR pathway.     

Modulation of oncogene expression by epidermal growth factor and gamma-interferon in A431 squamous carcinoma cells

Epidermal growth factor (EGF) acts, in a dose dependent manner, as both a mitogen and an inhibitor of growth of the A431 squamous carcinoma cell line. gamma-interferon (IFN) also inhibits A431 cell growth. The dual effects of EGF on A431 growth and expression of the oncogenes, EGF receptor (EGFR) and Ha-ras, were evaluated with or without gamma-IFN. A mitogenic level (10pM) of EGF had no effect on expression of EGFR 10 kb mRNA or protein. gamma-IFN combined with 10pM EGF caused an initial drop in EGFR mRNA not reflected at the protein level; at 72 hours, the level of EGFR 10kb mRNA rose and inhibition of cell growth was observed. Treatment with a cytostatic amount (10nM) of EGF resulted in decreased expression of EGFR 10kb mRNA and protein within 24 hours; combined treatment with gamma-IFN caused rapid cell death. Expression of Ha-ras mRNA paralleled that of EGFR mRNA upon treatment with 10pM EGF and/or gamma-IFN, but differed with 10nM EGF.     

Liver epithelial cell migration induced by epidermal growth factor or transforming growth factor alpha is associated with changes in the gene expression of secreted proteins

Summary Rat liver epithelial cells are induced to migrate by epidermal growth factor (EGF) or transforming growth factor alpha (TGF-α) in serum-free medium supplemented with insulin. Immunohistological staining of the migration tracks containing laminin and fibronectin has allowed a quantitative analysis of the process. The growth factor-induced migration is relatively slow, but very efficient. Between 24 and 48 h after exposure to EGF (or TGF-α), 50 to 70% of the cells have migrated away from their site of initial attachment and spreading. This delayed effect of the interaction of the receptor with its ligands is associated with changes in gene expression, but is not associated with a stimulation of cell proliferation. In serum-free medium supplemented with insulin, the cells secrete six major proteins, as revealed by SDS-polyacrylamide gel electrophoresis. The media of cultures supplemented with insulin plus EGF (or TGF-α) contain in addition two new proteins and an increased amount of fibronectin. One secreted protein is synthesized in significantly reduced amounts. The most conspicuously EGF-induced protein (EIP-1, Mr 47 000) is detected within 2 h, depends on the continued presence of the growth factor, and has not been detected as bound to the substratum. The stringent regulation of EIP-1 suggests that this gene product might participate in the modulation of the changes induced by the growth factor. The system is being used for the further analysis of the regulation of gene expression by EGF and of the migration of normal and neoplastically transformed epithelial cells. This paper is dedicated to the memory of Dr. Luis F. Leloir. A preliminary communication has been presented at the Cold Spring Harbor Meeting on Liver Gene Expression, May 1987. Editor's Statement Mitogen-stimulated gnees are an active area of study with fibroblastic systems. In this paper the approach is extended to epithelial cells and functional correlations are also made.     

Pioglitazone, a synthetic ligand for PPARγ, induces apoptosis in RB-deficient human colorectal cancer cells

No published data are available about the expression of peroxisome proliferator-activated receptor γ (PPARγ) and the role of PPARγ in retinoblastoma protein (RB)-deficient human colorectal cancer (CRC) cells (SNU-C4 and SNU-C2A). Our aim was to investigate whether PPARγ is expressed in SNU-C4 and SNU-C2A cells and to elucidate possible molecular mechanisms underlying the effect of pioglitazone, a synthetic ligand for PPARγ, on cell growth in these cell lines. RT-PCR and Western blot analysis showed that both human CRC cell lines expressed PPARγ mRNA and protein. Pioglitazone inhibited the cell growth of both cell lines through G2/M phase block and apoptosis. In addition, pioglitazone caused a down-regulation of the X chromosome-linked inhibitor of apoptosis (XIAP), Bcl-2, and cyclooxygenase-2 (COX-2) under conditions leading to PPARγ down-regulation. These results suggest that pioglitazone may have therapeutic relevance or significance in the treatment of human CRC, and the down-regulation of XIAP, Bcl-2, and COX-2 may contribute to pioglitazone-induced apoptosis in these and other RB-deficient cell lines and tumors.     

Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation

Pancreatic cancer is characterized by aggressive local invasion and early metastasis formation. Active migration of the pancreatic cancer cells is essential for these processes. We have shown previously that the pancreatic cancer cells lines CFPAC1 and IMIM-PC2 show high migratory activity, and we have investigated herein the reason for this observation. Cell migration was assessed using a three-dimensional, collagen-based assay and computer-assisted cell tracking. The expression of receptor tyrosine kinases was determined by flow-cytometry and cytokine release was measured by an enzyme-linked immunoassay. Receptor function was blocked by antibodies or pharmacological enzyme inhibitors. Both cells lines express the epidermal growth factor receptor (EGFR) as well as its family-member ErbB2 and the platelet-derived growth factor receptor (PDGFR)α, whereas only weak expression was detected for ErbB3 and no expression of PDGFRβ. Pharmacological inhibition of the EGFR or ErbB2 significantly reduced the migratory activity in both cell lines, as did an anti-EGFR antibody. Interestingly, combination of the latter with an anti-PDGFR antibody led to an even more pronounced reduction. Both cell lines release detectable amounts of EGF. Thus, the high migratory activity of the investigated pancreatic cancer cell lines is due to autocrine EGFR activation and possibly of other receptor tyrosine kinases.     

嵌合表皮生长因子疫苗E5T-mSEA的设计及抑瘤活性检测

表皮生长因子受体(Epidermal growth factor receptor,EGFR)及其配体在多种肿瘤细胞中高表达,免疫注射表皮生长因子(Epidermal growth factor,EGF)-载体蛋白所产生的抗体能够干扰EGFR与EGF的相互作用,进而抑制肿瘤生长。本实验室设计了EGF和TGFα的嵌合配体E5T,并将具有免疫增强作用的葡萄球菌肠毒素A(Staphylococcal enterotoxin A,SEA)与之融合。融合蛋白在大肠杆菌内表达后,通过金属螯合亲和层析,得到了纯度较高的E5T-mSEA融合蛋白。将E5T-mSEA免疫小鼠,能够产生高滴度的抗体,该抗体能够同时识别EGF和TGFα。将抗E5T-mSEA免疫血清与肿瘤细胞共培养,在1:10的稀释度下能显著抑制EGFR阳性肿瘤细胞的生长,而对EGFR阴性肿瘤细胞293T的生长没有明显影响。     

Platelet-derived growth factor requires epidermal growth factor receptor to activate p21-activated kinase family kinases

The platelet-derived growth factor (PDGF) receptor (PDGFR) transactivates the epidermal growth factor (EGF) receptor (ErbB1) to stimulate the cell migration of fibroblasts through an unknown mechanism (Li, J., Kim, Y. N. & Bertics, P. (2000) J. Biol. Chem. 275, 2951-2958). In this paper we provide evidence that the transactivation of the EGF receptor (EGFR) by PDGFR is essential for PDGF to activate p21-activated kinase (PAK) family kinases. Fetal calf serum (10%) transiently stimulates the PAK activity in NIH 3T3 fibroblasts. The activation of PAK was completely inhibited by either PDGFR-specific inhibitor (AG1295) or EGFR-specific inhibitor (AG1478), suggesting that serum requires either the PDGF- or EGF-dependent pathway or the combination of both to activate PAK. PDGF-induced activation of PAK is completely inhibited by either AG1295 or AG1478, indicating that PDGF requires both PDGFR and EGFR for PAK activation. In support of this notion, a mouse embryo fibroblast cell line derived from the EGFR -/- mouse (from Dr. Erwin Wagner) doesn't activate PAK in response to PDGF. Expression of human EGFR in this cell line restores the ability of the PDGF to induce PAK activation. Our results indicate that PDGF activates PAK through transactivation of ErbB1.     

Regulation of the epidermal growth factor receptor gene expression in a morphological variant isolated from an epidermal growth factor receptor-deficient small cell lung carcinoma cell line

Most cell lines derived from small cell lung carcinoma grow in an anchorage-independent manner; they neither possess epidermal growth factor binding activity nor express epidermal growth factor receptor (EGFR) mRNA. A variant AD320, which grew in an anchorage-dependent manner with altered morphology, was isolated from the small cell lung carcinoma cell line Lu134 by treatment with the demethylating agent 5-azacytidine. The analysis, using methylation-sensitive restriction enzymes, revealed that the methylation pattern was altered only in the structural region of the EGFR gene; EGFR mRNA and epidermal growth factor binding activity could be detected in the variant. In addition, drastic changes in gene expression including a decrease of creatine kinase B mRNA and an increase of c-myc mRNA were observed. The EGFR in the variant appeared to be an active part of the transmembrane signaling machinery since c-fos and c-jun mRNA accumulated after epidermal growth factor treatment, followed by EGFR and c-myc mRNA accumulation. A potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, also induced EGFR mRNA. Thus, the inducible regulatory mechanism for the EGFR gene was activated in the variant even though the EGFR gene was constitutively expressed.