In vivo evaluation of epidermal growth factor (EGF) receptor density on human tumor xenografts using radiolabeled EGF and anti-(EGF receptor) mAb 425

 In order to study the potential of non-invasive scintigraphic evaluation of the epidermal growth factor (EGF) receptor status in vivo, the biokinetics and tumor binding of ¹²⁵I-EGF and anti-(EGF receptor) mAb 425 were investigated in nude mice bearing human tumor xenografts with different EGF-receptor densities as determined by a radioreceptor assay. The results demonstrated a tumor uptake for both substances depending on the receptor level. The EGF receptor status, however, was reflected slightly better by the binding of EGF to tumor tissue compared to the mAb. The rapid blood clearance of EGF with a plasma half-life of less than 1 min led to a tumor-to-blood ratio of approximately 3 within 6 h after injection in tumors with a high receptor expression. A similar ratio for the mAb was not obtained before day 6 after injection. The absolute concentration of EGF, however, was low compared to the mAb. Therefore, it can be concluded that the EGF receptor status as a target for (radio)immunotherapy can be evaluated in vivo with EGF labeled with a short-life positron-emitting radionuclide or with monoclonal antibodies to the EGF receptor or their fragments. Received: 14 September 1995 / Accepted: 6 December 1995     

EGF receptor inhibitors increase ErbB3 mRNA and protein levels in breast cancer cells

The potential benefits of drugs directly targeting the ErbB receptors for cancer therapy have led to an extensive development within this field. However, the clinical effects of ErbB receptor-targeting drugs in cancer treatment are limited due to a high frequency of resistance. It has been reported that, when inhibiting the epidermal growth factor receptor (EGFR) with the tyrosine kinase inhibitor gefitinib, increased activation of ErbB3 via MET, or by re-localization of ErbB3 mediates cell survival. Here we show further evidence that members of the ErbB receptor family facilitate resistance to EGFR inhibitor treatment in ErbB2 overexpressing breast cancer cells. We found that gefitinib treatment increased ErbB3 expression, both at protein and mRNA levels. ErbB3 expression was upregulated not only by gefitinib but also by a panel of different EGFR inhibitors, suggesting that inhibition of EGFR in general affects ErbB3 expression. In addition, we found that gefitinib treatment increased ErbB2 expression levels while EGFR inhibitors decreased the activity of ErbB2. Concentrations of gefitinib that decreased phospho-ErbB2 reversely increased ErbB3 levels. We further examined changes induced by gefitinib treatment on mRNA levels of the most common genes known to be involved in breast cancer. As expected, we found that gefitinib downregulated genes whose functions were linked to cellular proliferation, such as Ki-67, topoisomerase II alpha and cyclins, and surprisingly downregulated gene expression of FAS which is involved in apoptotic signaling. Together, our data strongly suggest that resistance to EGFR inhibitors may result from the compensation of other family members and that combinations of anti-cancer drugs are required to increase the sensitivity of these treatments.     

A novel effect of EGF on mRNA stability

Expression of the epidermal growth factor (EGF) receptor gene is stimulated by EGF and the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). PMA elevates EGF receptor mRNA levels in human KB epidermoid carcinoma cells, but does not significantly affect the half-life of this mRNA when its decay is examined after the addition of actinomycin D. In contrast, EGF greatly prolongs the half-life of EGF receptor mRNA suggesting a possible mechanism for the stimulatory effect of EGF on EGF receptor mRNA levels. EGF also stabilizes beta-tubulin and beta-actin mRNAs but has very little effect on the degradation of total mRNA.     

Interferon gamma-induced EGF receptor transactivation depends on the level of expression of EGF receptor in epithelial cells

Recently, we demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to interferon γ (IFNγ) in epidermoid carcinoma A431 cells. It was shown that IFNγ-induced EGFR transactivation is impossible in the some cancer epithelial cells. Here, we hypothesize that IFNγ-dependent EGFR transactivation in these cells correlates to the amount of EGFR on the surface of the cell. To test this suggestion, a line of stably transfected HEK293 cells (HEK293Δ99 cells) expressing a high level of mutant EGFR that lacked 99 C-terminal residues was obtained. Unlike the parent HEK293 cells, which lacked transactivation, HEK293Δ99 cells demonstrated EGFR transactivation in response to IFNγ. In HEK293Δ99 and A431 cells, the time courses of EGFR activation induced by IFNγ have the same pattern. In HEK293Δ99, as in A431 cells, IFNγ-induced EGFR transactivation requires EGFR kinase activity and occurs via an autophosphorylation mechanism. Taken together, these data provide direct evidence for the dependence of IFNγ-induced EGFR transactivation upon EGFR expression level in epithelial cells.     

雌激素拮抗剂对胎盘EGF受体及其基因表达的影响

中期孕鼠在他莫昔芬作用下,其颌下腺,血清中ＥＧＦ含量下降,胎盘中ＥＧＦ受体结合位点数下降以及它的ｍＲＮＡ表达受到抑制,再次证实了他莫昔芬抑制雌激素诱导ＥＧＦ受体ｍＲＮＡ的表达。从而使ＥＧＦ受体结合位点数减少,因此,他莫昔芬对孕鼠胚胎生长发育有不可忽视的影响。     

Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization.

Binding of epidermal growth factor (EGF) to its receptor results in a cascade of events that culminate in cell division. The receptor is present on the cell surface in two forms of high and low affinity binding for EGF. EGF binding activates the receptor's intracellular tyrosine kinase activity and subsequently causes the receptor to be rapidly internalized into the cell via clathrin-coated pits. We have cloned the EGF receptor cDNA into a retroviral expression vector and made mutations in vitro to investigate the function of different receptor domains. Deletion of cytoplasmic sequences abolishes high but not low affinity sites as well as impairing the ability of the protein to internalize into cells. Thus, cytoplasmic sequences must be involved in the regulation of high affinity sites and are required for EGF-induced receptor internalization. A four amino acid insertion mutation at residue 708 abolishes the protein-tyrosine kinase activity of the immunoprecipitated receptor. However, this receptor mutant exhibits both the high and low affinity states, internalizes efficiently and is able to cause cells to undergo DNA synthesis in response to EGF. Another four amino acid insertion mutation (residue 888) abolishes protein-tyrosine kinase activity, high affinity binding, internalization and mitogenic responsiveness. Finally, a chimaeric receptor composed of the extracellular EGF binding domain and the cytoplasmic v-abl kinase region transforms Rat-I cells. This chimaeric receptor possesses intrinsic protein tyrosine kinase activity which cannot be regulated by EGF. Moreover, EGF fails to induce the internalization of the chimaeric receptor.     

Role of EGF receptor tyrosine kinase activity in antiapoptotic effect of EGF on mouse hepatocytes

The apoptotic Fas pathway is potentially involved in the pathogenesis of liver diseases. Growth factors, such as epidermal growth factor (EGF), can protect cells against apoptosis induced by a variety of stimuli, including Fas receptor stimulation. However, the underlying mechanisms of this protection have yet to be determined. We investigated the involvement of EGF receptor (EGFR) tyrosine kinase (TK) activity in the antiapoptotic effect of EGF on primary mouse hepatocyte cultures. Cells undergoing apoptosis after treatment with anti-Fas antibody were protected by EGF treatment. This protection was significantly but partially decreased when cells were treated with two specific inhibitors of the TK activity of EGFR. Evaluation of the efficacy of these compounds indicated that they were able to abolish EGFR autophosphorylation and postreceptor events such as activation of mitogen-activated protein kinases and the phosphatidylinositol 3'-kinase pathways as well as increases in Bcl-x(L) mRNA and protein levels. This leads us to postulate that EGF exerts its antiapoptotic action partially through the TK activity of EGFR. In addition, our results suggest that Bcl-x(L) protein upregulation caused by EGF is linked to the TK activity of its receptor.     

TGF-alpha-driven tumor growth is inhibited by an EGF receptor tyrosine kinase inhibitor.

The simultaneous presence of the EGFR and its ligand TGF-alpha in human tumor tissues suggests that autocrine TGF-alpha stimulation drives tumor growth. Here we show that autocrine TGF-alpha stimulation does cause increased tumor growth in vivo, an effect that was proven to be mediated via EGFR activation, and that this TGF-alpha/EGFR autocrine loop was accessible to an EGFR specific tyrosine kinase inhibitor. Clones of the EGFR expressing glioma cell line U-1242 MG were transfected with TGF-alpha cDNA using a tetracycline-inhibitory system for gene expression. TGF-alpha expression was inhibited by the presence of tetracycline, and subcutaneous tumors forming from cell lines injected into nude mice could be inhibited by feeding mice tetracycline. We confirmed that TGF-alpha mRNA and protein were present in these tumors and that, subsequently, the endogenous EGFR was activated. Tumor growth could be inhibited by an EGFR specific tyrosine kinase inhibitor of the type 4-(3-chloroanilino)-6,7-dimethoxy-quinazoline, administered daily by intraperitoneal injection, thereby interrupting the autocrine loop.     

Effect of hCG administration at the onset of oestrus on early embryo survival and development in Meishan gilts

A study was designed to advance the time of the ovulatory luteinizing hormone (LH) surge in Meishan gilts by human chorionic gonadotrophin (hCG) administration at the onset of oestrus and assess the effect on embryo survival and development. Twelve Meishan gilts were observed six times daily for oestrous behaviour and bred at 24, 36 and 48 h after observed oestrous onset. Six of those gilts were administered an ovulatory dose of hCG (500 IU) at observed oestrous onset. Blood samples were collected at oestrous onset (Day —2) and on Days 0, 2, 6, 9, 13, 16, 20, 23, 27 and 30 of gestation. All gilts were slaughtered on Day 30 of gestation and embryo survival and conceptus development assessed. Ovulation rate did not differ between control and hCG treated gilts (18.5 and 17.7 respectively; P>0.1) while the number of live conceptuses per gilt (17.2 and 12.8 respectively; P<0.08) and embryo survival rate (92.1 and 75.8% respectively; P<0.1) both tended to be reduced by the hCG treatment. Placental weight (17.2 and 23.1 g; P<0.01) was significantly increased in hCG treated gilts, while embryo weight (1.2 and 1.4 g; P<0.06) and placental length (42.8 and 47.2 cm; P<0.07) both tended to be increased in hCG treated gilts. Crown rump length (P>0.1) and allantoic fluid volume (P>0.1) did not differ between the treatment groups. Serum progesterone concentrations did not differ with treatment overall (P>0.1) but were significantly elevated (P< 0.05) at 48 h postoestrus in the hCG treated gilts compared to control gilts. Overall, these results indicate that advancing the time of the LH surge to oestrous onset, as in European breeds, compromised embryo survival and suggests that the longer time interval between oestrous onset and ovulation is important for the high rate of embryo survival in the Meishan pig.     

The EGF receptor provides an essential survival signal for SOS-dependent skin tumor development

The EGF receptor (EGFR) is required for skin development and is implicated in epithelial tumor formation. Transgenic mice expressing a dominant form of Son of Sevenless (SOS-F) in basal keratinocytes develop skin papillomas with 100% penetrance. However, tumor formation is inhibited in a hypomorphic (wa2) and null EGFR background. Similarly, EGFR-deficient fibroblasts are resistant to transformation by SOS-F and rasV12, however, tumorigenicity is restored by expression of the anti-apoptotic bcl-2 gene. The K5-SOS-F papillomas and primary keratinocytesfrom wa2 mice display increased apoptosis, reduced Akt phosphorylation and grafting experiments imply a cell-autonomous requirement for EGFR in keratinocytes. Therefore, EGFR functions as a survival factor in oncogenic transformation and provides a valuable target for therapeutic intervention in a broader range of tumors than anticipated.     

Effect of hCG administration on day 7 of the estrous cycle on follicular dynamics and cycle length in cows

The use of hCG in cattle at breeding or at different times after breeding has been associated with extension in estrous cycle length among cows that do not become pregnant. The objective of this study was to determine whether the increase in estrous cycle length observed in hCG-treated cows that fail to become pregnant is due to changes in ovarian follicular dynamics. Twelve nonbred lactating cows were randomly assigned either to receive hCG on Day 7 of the cycle (Day 0 = day of estrus, n = 6) or to serve as controls (n = 6). Ultrasound scanning was conducted daily from Day 0 until the onset of the next ovulation to monitor follicular and corpus luteum (CL) dynamics. Blood samples were collected for progesterone analysis at each ultrasound session. Ovulation of the Day 7 follicle occurred in all 6 hCG-treated cows. The time of emergence of the second-wave of follicular growth was advanced in hCG-treated cows but was not statistically different (P > 0.05) from that of the control cows (10.8 +/- 0.3 vs 12.7 +/- 1.4 d). The mean diameter of the second-wave dominant follicle from Days 15 to 18 was not different (P > 0.05) between the treatment groups. However, the second-wave dominant follicle had a slower growth rate (0.8 vs 1.3 mm/d) among cows treated with hCG compared with that of the controls. The second-wave dominant follicle was the ovulatory follicle in 5 control cows, but only in 3 hCG-treated cows. The dominant follicle from the third wave ovulated in 1 control and in 3 hCG-treated cows. The lifespan of the spontaneous CL and the time to low progesterone levels (< 1 ng/ml) were not different between the control and hCG-treated cows. These results suggest an altered follicular dynamic but no extension in estrous cycle length when hCG is administered on Day 7 of the cycle in postpartum cows.     

Variation in EGF-induced EGF receptor downregulation in human hepatoma-derived cell lines expressing different amounts of EGF receptor

The effect of epidermal growth factor (EGF) receptor overexpression on ligand-induced EGF receptor downregulation was examined using a hepatoma-derived cell line, PLC/PRF/5, which expresses normal amounts of the EGF receptor, and a subline, NPLC/PRF/5, which expresses 10-fold more receptors at its cell surface. PLC/PRF/5 cells efficiently downregulated surface receptor levels upon exposure to saturating and subsaturating concentrations of EGF; the rate of receptor downregulation corresponded to that of ligand-receptor internalization. Upon internalization, EGF receptors were degraded and receptor biosynthesis remained at basal levels. EGF surface receptor remained downregulated for as long as cells were exposed to EGF. By contrast, surface EGF receptor abundance in NPLC/PRF/5 cells decreased by only 5-15% after 1-4 h incubation with subsaturating doses of EGF and actually increased by 67% within 20 h. Exposure of these cells to saturating concentrations of EGF induced modest decreases in surface receptor abundance during the initial 12 h incubation, followed by a progressive decline to 30% of initial values by 24 h. Relative ligand-receptor internalization rates in NPLC/PRF/5 cells were lower than those in PLC/PRF/5, although their surface receptor population was even higher than that predicted by the decreased internalization rates. EGF receptor degradation in NPLC/PRF/5 cells was also inhibited; exposure to saturating levels of EGF for more than 16 h was necessary before significant degradation occurred. Receptor protein and mRNA biosynthesis in NPLC/PRF/5 were stimulated by 8 h exposure to EGF but when saturating concentrations of EGF were present for 16 h, receptor biosynthesis was inhibited. EGF receptor overexpression circumvents the downregulatory effect of EGF by decreasing the rate of receptor internalization, inhibiting degradation of the internalized receptor pool, and stimulating EGF receptor biosynthesis. Conversely, receptor downregulation becomes pronounced at late times when receptor degradation is high and biosynthesis is inhibited.     

EGF receptor activities in mammalian development

The receptor for epidermal growth factor (EGF) and its analog transforming growth factor alpha (TGF alpha) is ubiquitous, implying quite general roles for EGF/TGF alpha in cell viability and tissue maintenance in adult tissues. There is also evidence that the EGF receptor is active in promoting wound healing and tissue regeneration in adult organs, such as skin, liver, and intestinal epithelium. It is likely that EGF receptors have more specific roles during the gestation period. For example, we have detected EGF receptors on the 3.5-day blastocyst (trophectoderm) surface and since TGF alpha-like mRNA sequences and peptides have been detected at this time (Rappolee et al., Science 241:1823, 1988), there is a strong implication for autocrine stimulation in pre- and peri-implantation stage embryos. Paracrine stimulation between the embryo and maternal tissues is also likely since both receptors and TGF alpha are present in decidual cells. Therefore EGF receptors may take part in growth regulation of the early embryo and in the process of implantation. Other examples where EGF receptors may play specific roles during embryonic development are discussed.     

Negative co-operativity in the EGF receptor

Scatchard analyses of the binding of EGF (epidermal growth factor) to its receptor (EGFR) yield concave up Scatchard plots, indicative of some type of heterogenity in ligand-binding affinity. This was typically interpreted as being due to the presence of two independent binding sites: one of high affinity representing ≤10% of the receptor population, and one of low affinity making up the bulk of the receptors. However, the concept of two independent binding sites is difficult to reconcile with the X-ray structures of the dimerized EGFR that show symmetrical binding of the two ligands. A new approach to the analysis of 125I-EGF-binding data combined with the structure of the singly-occupied Drosophila EGFR have now shown that this heterogeneity is due to the presence of negative co-operativity in the EGFR. Concerns that negative co-operativity precludes ligand-induced dimerization of the EGFR confuse the concepts of linkage and co-operativity. Linkage refers to the effect of ligand on the assembly of dimers, whereas co-operativity refers to the effect of ligand binding to one subunit on ligand binding to the other subunit within a preassembled dimer. Binding of EGF to its receptor is positively linked with dimer assembly, but shows negative co-operativity within the dimer.