Modulation of mouse preimplantation development by epidermal growth factor receptor antibodies,antisense RNA,and deoxyoligonucleotides

Two-cell mouse preimplantation embryos were cultured for 48 h in four different reagents to modulate epidermal growth factor (EGF) receptor function. These were rabbit polyclonal and mouse monoclonal antibodies to EGF receptor, EGF receptor antisense RNA, and EGF receptor antisense deoxyoligonucleotides. Embryos were scored for two endpoints: onset of cavitation as a measure of trophectoderm differentiation and mean embryo cell number as a measure of cell proliferation. The consistent observations were that cavitation was significantly accelerated by antibodies and delayed by antisense RNA and antisense deoxyoligonucleotides. None of these reagents exerted a significant effect on mean embryo cell number, with one exception the polyclonal antibody. Our interpretation of these observations is that the antibody binding facilitated cavitation by mimicking natural ligand-receptor binding and inducing the signal transduction cascade that is typical for the EGF receptor. In the case of antisense RNA or deoxyoligonucleotide, we propose that they delayed onset of cavitation by interfering with EGF receptor production. We hypothesize that during this period of development, EGF receptor is concerned predominantly with the regulation of differentiation more than with cell proliferation. © 1993Wiley-Liss, Inc.     

EGF-R antisense RNA blocks expression of the epidermal growth factor receptor and suppresses the transforming phenotype of a human carcinoma cell line.

We have used an antisense approach to investigate the role of overexpression of the normal human epidermal growth factor (EGF) receptor in the transformed phenotype of KB cells, which are a tumor derived human cell line. Initial experiments performed in vitro, showed that antisense RNA complementary to the entire coding region (AS-FL) or to parts of the EGF-R mRNA (AS-3', AS-5', and AS-K) effectively blocked translation of EGF-R mRNA. In addition, upon microinjection into KB cells, the in vitro synthesized antisense RNAs were able to inhibit transiently the synthesis of EGF-R. Inhibition was concentration-dependent, both in vitro and in cells, and the most effective constructs were those complementary to the entire coding region (AS-FL) or to the 3'-coding end of the mRNA (AS-3'). Transfection of the same EGF-R antisense RNA constructs into the human epidermoid carcinoma KB cell line gave rise to several clones stably expressing elevated levels of antisense RNA and resulting in low residual levels of EGF receptor. The most reduced clones exhibited a totally restored serum-dependent growth and were severely impaired in colony formation and growth in agar. In addition the severity of the phenotype was directly proportional to the residual amount of EGF-R expressed. We conclude that over-expression of normal EGF-R plays a direct primary role in the development of the transformed phenotype of this human cancer cell line.     

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.     

The proliferative and morphologic responses of a colon carcinoma cell line (LIM 1215) require the production of two autocrine factors.

The role of autocrine growth factors in tumor cell growth has been difficult to prove. Our results indicate that more than one autocrine factor is required for the autonomous growth of the LIM 1215 colonic carcinoma cell line. Furthermore, the morphologic changes induced by epidermal growth factor (EGF) are also density dependent and appear to require a synergistic autocrine factor. The serum-free proliferation of the colonic carcinoma cell line LIM 1215 depends on cell density and the presence of EGF (A. Sizeland, S. Bol, and A.W. Burgess, Growth Factors 4:129-143, 1991). At cell densities below 10(4)/cm2, conditioned medium (from cells at a density of 10(5)/cm2) was required for the cells to elicit a mitogenic response to exogenous EGF. At higher cell densities (10(5)/cm2), the cells were independent of both exogenous EGF and conditioned medium. In addition, the EGF receptor was found to be phosphorylated on tyrosine in LIM 1215 cells proliferating at high density, suggesting that the autocrine production of transforming growth factor alpha (TGF alpha) and subsequent ligation to the EGF receptor was occurring. The proliferation of cells at high density was partly inhibited by TGF alpha antibodies but was almost completely inhibited by an antisense oligonucleotide to TGF alpha. The antisense inhibition could be overcome by the addition of EGF, indicating that the effect of the antisense TGF alpha oligonucleotide was on the production of autocrine TGF alpha. LIM 1215 cells were also observed to undergo morphologic changes (spreading and actin cable organization) in response to EGF. These changes were density dependent, but they occurred with a cell density dependence different from that of the proliferative response. These results suggest two possibilities: that the morphologic changes and proliferative responses have different sensitivities to the autocrine factors or that the actions of the autocrine factors are mediated through different signal transduction pathways.     

Structure and localization of genes encoding aberrant and normal epidermal growth factor receptor RNAs from A431 human carcinoma cells.

A431 cells have an amplification of the epidermal growth factor (EGF) receptor gene, the cellular homolog of the v-erb B oncogene, and overproduce an aberrant 2.9-kilobase RNA that encodes a portion of the EGF receptor. A cDNA (pE15) for the aberrant RNA was cloned, sequenced, and used to analyze genomic DNA blots from A431 and normal cells. These data indicate that the aberrant RNA is created by a gene rearrangement within chromosome 7, resulting in a fusion of the 5' portion of the EGF receptor gene to an unidentified region of genomic DNA. The unidentified sequences are amplified to about the same degree (20- to 30-fold) as the EGF receptor sequences. In situ hybridization to chromosomes from normal cells and A431 cells show that both the EGF receptor gene and the unidentified DNA are localized to the p14-p12 region of chromosome 7. By using cDNA fragments to probe DNA blots from mouse-A431 somatic cell hybrids, the rearranged receptor gene was shown to be associated with translocation chromosome M4.     

Effects of stimulation of AMP-activated protein kinase on insulin-like growth factor 1- and epidermal growth factor-dependent extracellular signal-regulated kinase pathway

AMP-activated protein kinase (AMPK) is tightly regulated by the cellular AMP:ATP ratio and plays a central role in the regulation of energy homeostasis. Previously, AMPK was reported to phosphorylate serine 621 of Raf-1 in vitro. In the present study, we investigated a possible role of AMPK in extracellular signal-regulated kinase (Erk) cascades, using 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), a cell-permeable activator of AMPK and antisense RNA experiments. Activation of AMPK by AICAR in NIH-3T3 cells resulted in drastic inhibitions of Ras, Raf-1, and Erk activation induced by insulin-like growth factor 1 (IGF-1). Expression of an antisense RNA for the AMPK catalytic subunit decreased the AMPK activity and significantly diminished the AICAR effect on IGF-1-induced Ras activation and the subsequent Erk activation, indicating that its effect is indeed mediated by AMPK. Phosphorylation of Raf-1 serine 621, however, was not involved in AMPK-mediated inhibition of Erk cascades. In contrast to IGF-1, AICAR did not block epidermal growth factor (EGF)-dependent Raf-1 and Erk activation, but our results demonstrated that multiple Raf-1 upstream pathways induced by EGF were differentially affected by AICAR: inhibition of Ras activation and simultaneous induction of Ras-independent Raf activation. The activities of IGF-1 and EGF receptor were not affected by AICAR. Taken together, our results suggest that AMPK differentially regulate Erk cascades by inhibiting Ras activation or stimulating the Ras-independent pathway in response to the varying energy status of the cell.     

Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA

The epidermal growth factor (EGF) receptor plays a key role in the control cellular proliferation, and its homology to the avian erythroblastosis virus erb B oncogene implicates its involvement in cellular transformation. The establishment of a correlation between the various structural domains of the EGF receptor and their functional counterparts would greatly advance our understanding of these processes. To this end, we have constructed an expression vector containing the SP6 viral promoter and an adjacent cDNA fragment encoding the full-length EGF receptor. Upon addition of SP6 RNA polymerase, this DNA is capable of generating large amounts of EGF receptor mRNA; this RNA can then be translated in vitro into immunoprecipitable EGF receptor protein. The translational efficiency of this EGF receptor RNA was found to be relatively low: approx. 100-fold lower than globin RNA synthesized using SP6 RNA polymerase. Use of these tools should now permit the synthesis and analysis of mutated EGF receptor protein in an effort to clarify the role of this receptor in growth control.     

Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells

Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.     

Epidermal growth factor (EGF) stimulates EGF receptor synthesis

Epidermal growth factor (EGF) binds to the extracellular domain of a specific 170,000-dalton transmembrane glycoprotein; this results in rapid removal of both ligand and receptor from the cell surface. In WB cells, a rat hepatic epithelial cell line, ligand-directed receptor internalization leads to receptor degradation. We tested whether the EGF receptor was replenished at a constitutive or enhanced rate following EGF binding by immunoprecipitating biosynthetically labeled EGF receptor from cells cultured with [35S]methionine. EGF stimulated receptor synthesis within 2 h in a dose-dependent manner; this was particularly evident when examining the nascent form of the receptor. To determine the site of EGF action, total WB cell RNA was transferred to nitrocellulose paper after electrophoresis and was hybridized to cDNA probes from both the external and cytoplasmic coding regions of the human EGF receptor. EGF increased receptor mRNA by 3-5-fold. Therefore, at least in some cells, the surface action of EGF that leads to EGF receptor degradation is counterbalanced by a positive effect on receptor synthesis.     

Modulation of the epidermal growth factor receptor by the human papillomavirus type 16 E5 protein in raft cultures of human keratinocytes

It has been shown that the E5 protein of the human papillomavirus type 16 modulates epidermal growth factor receptor downregulation in monolayer cultures of human keratinocytes and mouse fibroblasts. We have now analysed the effect of this protein on the expression, the distribution and the activation of EGF receptors in raft cultures derived from an E5-transfected human keratinocyte cell line. The epithelia generated in these cultures were stratified and exhibited suprabasal expression of cytokeratins 1 and 10, which are known markers of early epidermal differentiation. In situ hybridization with an antisense riboprobe to the human papilloma virus type 16 E5 protein revealed a homogeneous gene expression within the entire epithelium of E5-transfected but not empty vector-transfected control cultures. Treatment of serum-starved rafts with EGF for 48 hours led to a strong decrease of suprabasal EGF receptors in control cultures, but not in rafts of E5-expressing cells. Under these conditions, no activated receptors were observed in control cultures, but activated receptors were still present in E5-raft cultures. Our results indicate that human papilloma virus type 16 E5-mediated modulation of EGF receptor expression occurs in a time- and structure-dependent manner in epithelial equivalents of human keratinocytes.     

Inhibition of the androgen receptor by antisense oligonucleotides regulates the biological activity of androgens in SZ95 sebocytes.

One novel strategy for the blockade of the androgen receptor could be the selective inhibition of androgen receptor by antisense oligonucleotides or small interfering RNA molecules. Here we describe the down regulation of the androgen receptor in cultured human SZ95 sebocytes with antisense oligonucleotides modified with phosphorothioates and 2'- O-methylribosyl residues. The ability of antisense oligonucleotides to cross the cellular membrane was enhanced by establishing a transient transfection system based on cationic lipid vesicles. Both antisense oligonucleotide types administered caused assumedly translational arrest. Dose-dependent inhibition of androgen receptor protein expression was observed after SZ95 sebocyte transfection with modified phosphorothioate oligonucleotides and modified 2'- O-methylribonucleotides which were directed against the translational start of the androgen receptor mRNA. The strongest transient inhibition of androgen receptor expression was detected after 14 hours with 1.0 muM antisense 2'- O-methylribonucleotides (88+/-1.3%, p<0.001). With longer recovery times than 24 hours, androgen receptor protein expression returned to the native control levels. Inhibition of the expression of androgen receptor by antisense oligonucleotides, reduced the enhanced proliferation of SZ95 sebocytes challenged by testosterone and 5alpha-dihydrotestosterone. This administration opens new therapeutic possibilities in androgen-associated skin diseases, since we could also show androgen inhibition with these antisense oligonucleotides in a reconstituted human epidermis model (Horm Metab Res 2007; 39:157-165).     

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.