Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts

Cellular insulin-like growth factor binding protein-3 (IGFBP-3) mRNA and IGFBP-3 levels in conditioned medium were consistently higher in cultures of late passage normal (old) fibroblasts and prematurely senescent fibroblasts derived from Werner syndrome (WS) during quiescence induced by serum depletion and during the renewed growth ensuing after serum repletion, compared to cultures of early passage normal (young) fibroblasts. Molar ratios of IGFBP-3/IGF-II were always higher in senescent cultures and maintained a hierarchy of old > WS > young human diploid fibroblasts. Transfection into fibroblasts of the normal full-length IGFBP-3 cDNA in an expression vector resulted in a significant reduction in colony formation compared to cells transfected with an empty expression vector (no cDNA) or with IGFBP-3 cDNA altered by a 273 base pair (bp) deletion. Addition to old and young cultures of recombinant human IGFBP-3 and IGF-I at 1:1 or 5:1 molar ratios inhibited IGF-I-mediated DNA synthesis by approximately 70-80%. These data indicate that IGFBP-3 may play an important role in the quiescent and senescent growth arrest of HDF.     

Time-dependent alteration of epidermal growth factor receptor in rat stomach by ethanol feeding

This study investigated the time-dependent effects of ethanol (EtOH) feeding on epidermal growth factor binding and epidermal growth factor-mediated functions in the stomach. Adult male rats were fed either an isocaloric control or EtOH-containing liquid diet (36% total calories as EtOH) for 2, 4 and 6 weeks. At the end of each feeding period, animals were sacrificed and the stomach was dissected for the sample preparation. EtOH caused a time-dependent alteration (r = 0.89) of the 125I-epidermal growth factor binding to the gastric mucosal membrane (% control: week 2, 114%; week 4, 64%* and week 6, 45%*, n = 5, *P < 0.05). Protein kinase analysis also showed that EtOH caused a time-dependent decrease of epidermal growth factor-stimulated autophosphorylation of epidermal growth factor receptor protein (180 kDa) during three feeding periods. Western blot analysis, using anti-tyrosine phosphorylated epidermal growth factor receptor (active form) antibody, revealed a major immunoreactive protein band (180 kDa) in all samples pre-incubated with 1 microM epidermal growth factor. Consistent with data from kinase analysis, treatment of EtOH decreased the immunoreactivity of the active form of epidermal growth factor receptor (180 kDa) in the stomach. In conclusion, EtOH feeding caused a time-dependent alteration of epidermal growth factor receptor in the stomach, which may be one of the mechanisms underlying the gastric pathology associated with alcohol abuse.     

Intestinal adaptation is enhanced by epidermal growth factor independent of increased ileal epidermal growth factor receptor expression

BACKGROUND/PURPOSE: Intestinal adaptation after massive small bowel resection (SBR) is augmented by epidermal growth factor (EGF) via an unknown mechanism. We recently have observed that EGF increases the expression of EGF receptor mRNA and protein content in the remnant ileum after SBR. The purpose of this study was to determine whether the magnitude of EGF-induced receptor expression correlates with intestinal adaptation. METHODS: A 50% proximal SBR or sham operation (bowel transection with reanastomosis) was performed on male ICR mice. Animals from each group were then selected randomly to receive either human recombinant EGF (150 microg/kg/d) or saline by twice daily intraperitoneal injections. The remnant ileum was harvested at 1 week, and parameters of adaptation measured as changes in protein content. Ileal EGF receptor mRNA was quantitated using a ribonuclease protection assay. Changes in the expression ileal EGF receptor protein were determined by Western blot after immunoprecipitation. Comparisons of mean values between groups was performed using analysis of variance (ANOVA) and a P value of less than .05 was considered significant. Values are presented as mean +/- SEM. RESULTS: EGF was mitogenic to the ileum after sham operation as monitored by increases in ileal protein content (2.21 +/- 0.002 mg/cm Sham v 2.97 +/- 0.25 mg/cm Sham +/- EGF; P < .05). After SBR, adaptation resulted in increased ileal protein content (4.45 +/- 0.27 mg/cm), which was substantially boosted by EGF (5.98 +/- 0.39 mg/cm; P < .05). No differences were detected in ileal EGF receptor mRNA or protein expression between Sham or SBR groups that did not receive EGF. However, EGF significantly enhanced the expression of ileal EGF receptor mRNA to an equal extent after both sham and SBR (approximately threefold). The magnitude of this increase in EGF receptor protein (four- to sixfold) was similar in both EGF groups as shown by Western blotting. CONCLUSIONS: Changes in ileal EGF receptor expression are not mandatory for adaptation to occur. EGF upregulates the expression of mRNA and protein for its own intestinal receptor in vivo. Because EGF-induced receptor expression was comparable after both SBR and Sham operation, the beneficial effect of EGF during adaptation is likely caused by other factors in addition to increased receptor expression.     

Enhanced expression of the protooncogenes c-myc and c-fos in normal and malignant renal growth

The protooncogenes c-myc and c-fos play an important role in growth and differentiation of renal tissue. They are highly expressed during embryogenesis in the mitotically active tubule epithelium, while in terminally differentiated tubule cells of the kidney the expression is completely shut off. Furthermore, induction of cell proliferation in cultured renal cells by addition of growth factors is preceded by enhanced expression of c-myc and c-fos. Increased expression of these protooncogenes is also obtained by treatment of kidney cells in culture with the potent nephrocarcinogen N-dimethylnitrosamine and also with the nephrotoxin and possibly nephrocarcinogen S-(1,2-dichlorovinyl)-L-cysteine. Finally, the expression of c-myc and c-fos is induced after unilateral nephrectomy during compensatory renal growth in the remaining kidney and also during regenerative cell proliferation after in vivo application of the strong nephrotoxins folic acid and mercury chloride.     

Regulation of expression of epidermal growth factor receptors in gonadotropes by epidermal growth factor and estradiol: studies in cycling female rats

Changes in expression of epidermal growth factor (EGF) receptors by gonadotropes parallel those of GnRH receptors. Gonadotropes increase their expression of EGF receptors (EGFR) during diestrus to reach a peak on the morning of proestrus. This is followed by a decline in expression to reach a nadir by estrus. We hypothesized that regulatory factors that stimulate changes in GnRH receptors might mediate the same changes in EGFR. To test this hypothesis, pituitary cells were collected from cycling rats and grown overnight in media with or without serum, 100 pM estradiol, or 60 ng/ml activin. On the next day, some of the cultures were further stimulated with 1 nM GnRH (4 h). The cells were then dual-labeled for EGFR and LHbeta or FSHbeta antigens and analyzed for their content of EGFR and gonadotropins. Neither activin nor estradiol increased percentages of cells with gonadotropin antigens and EGFR. Estradiol decreased percentages of cells with EGFR and LH in proestrous rats and those with EGFR and FSH in diestrous rats. The estradiol-mediated decline in EGFR expression during proestrus is similar to that seen when GnRH receptors are studied. Serum containing media alone increased percentages of LH and FSH cells with EGFR in populations from estrous or metestrous rats. Therefore, further experiments were conducted to learn if serum factors or EGF might be a regulator. Removal of serum from the growth media did not prevent the increase in percentages of LH cells with EGFR over the 18-h growth period. However, removal of serum did prevent the increased percentages of FSH cells with EGFR. Similarly, adding 1:100 anti-EGF to the serum containing media did not affect expression of EGFR by LH cells. However, it did cause a 27% decrease in percentages of FSH cells with EGFR. Finally, when 10 ng/ml EGF was added to metestrous populations in serum-free media there was a 1.4-1.5-fold increase in percentages of LH or FSH cells with EGFR. Collectively, these studies show that EGF receptors are not stimulated in gonadotropes by the same hormones that up-regulate GnRH receptors. Furthermore, EGF itself may be among the factors that up regulate EGFR in gonadotropes. EGF receptors may be down-regulated by estradiol during proestrus, but the effect is limited to LH cells. Finally, EGF's differential effects on LH and FSH cells suggests that it may selectively act on monohormonal gonadotropes. EGF receptors may be a marker for a unique subset of developing gonadotropes.     

Antisense estrogen receptor RNA expression increases epidermal growth factor receptor gene expression in breast cancer cells

In human breast cancer, progression to a more malignant phenotype is often accompanied by decreased expression of estrogen receptor (ER) and increased expression of epidermal growth factor receptor (EGFR). Higher levels of this receptor tyrosine kinase are found in tumors lacking ER, and a quantitative, inverse relationship exists between the level of ER and EGFR mRNA in human breast cell lines. Antisense ER (ASER) RNA was used to evaluate the consequence of decreased ER expression in breast cancer cells, specifically to determine whether ER is involved in the regulation of EGFR gene expression. ER-positive MCF-7 human breast cancer cells were transfected with ASER, and clones constitutively expressing ASER RNA had decreased ER and up to a 3-fold increase in the expression of EGFR mRNA. To confirm that this observation was a direct consequence of ASER expression, a metal-inducible ASER expression construct was transfected into MCF-7 cells, and transfected clones were isolated and characterized. Northern analysis revealed an induction of ASER RNA within 1 h of the addition of zinc, which was followed by a 4-fold increase in EGFR mRNA levels, maximal at 6-12 h. The basal level of expression of the glucocorticoid receptor is also inversely related to that of ER among breast cancer cell lines, but neither constitutive nor inducible expression of ASER affected the expression of glucocorticoid receptor. These data support the hypothesis that the level of expression of ER specifically influences the expression of EGFR in human breast cancer cells and provides a potential link between loss of steroid sensitivity and the acquisition of autonomous growth.     

Suppression of postischemic hippocampal nerve growth factor expression by a c-fos antisense oligodeoxynucleotide

We examined the uptake and distribution of an antisense phosphorothioated oligodeoxynucleotide (s-ODN) to c-fos, rncfosr115, infused into the left cerebral ventricle of male Long-Evans rats and the effect of this s-ODN on subsequent Fos, NGF, neurotrophin-3 (NT-3), and actin expression. To establish the uptake and turnover of s-ODN in the brain, we studied the copurification of the immunoreactivity of biotin with biotinylated s-ODN that was recovered from different regions of the brain. A time-dependent diffusion and the localization of s-ODN were further demonstrated by labeling the 3'-OH terminus of s-ODN in situ with digoxigenin-dUTP using terminal transferase and detection using anti-digoxigenin IgG-FITC. Cellular uptake of the s-ODN was evident in both the hippocampal and cortical regions, consistent with a gradient originating at the ventricular surface. Degradation of the s-ODN was observed beginning 48 hr after delivery. The effectiveness of c-fos antisense s-ODN was demonstrated by its suppression of postischemic Fos expression, which was accompanied by an inhibition of ischemia-induced NGF mRNA expression in the dentate gyrus. Infusion of saline, the sense s-ODN, or a mismatch antisense s-ODN did not suppress Fos expression. That this effect of c-fos antisense s-ODN was specific to NGF was demonstrated by its lack of effect on the postischemic expression of the NT-3 and beta-actin genes. Our results demonstrate that c-fos antisense s-ODN blocks selected downstream events and support the contention that postischemic Fos regulates the subsequent expression of the NGF gene and that Fos expression may have a functional component in neuroregeneration after focal cerebral ischemia-reperfusion.     

Induction of neurofilament triplet proteins in PC12 cells by nerve growth factor

The localization of neurofilament triplet proteins in PC12 cells grown in the absence of (PC12-) or maintained in the presence of (PC12+) nerve growth factor (NGF) was studied using indirect immunofluorescence and monospecific, immunosorbent purified antibodies to 68,000 (P68), 150,000 (P150) and 200,000 (P200) dalton neurofilament proteins. The intensity of immunofluorescent staining of the triplet proteins was always greater in PC12+ compared with PC12-cells. Neuritic staining was seen in PC12+ cells with all 3 monospecific antibodies to neurofilament proteins. However, the perikaryal distribution of each of the neurofilament proteins differed in both PC12+ and PC12-cells. Monospecific antibodies to P68 protein yielded a 'ball-like' cytoplasmic staining pattern whereas monospecific antibodies by P150 protein stained in a stippled pattern. Monospecific antibodies to P200 on the other hand diffusely stained the perikaryal cytoplasm with very faint but detectable foci of 'ball-like' configurations and stippling. Electron microscopic study of PC12+ and PC12-cells revealed intermediate filaments in the cell bodies of both as well as in the processes of the former. 'Ball-like' clusters of such filaments were rarely seen. However, these filaments lacked the three-dimensional organization typical of intact neurofilaments. It is concluded that PC12 cells contain dissociated or incompletely assembled immunoreactive neurofilament triplet proteins and that these proteins can be induced by NGF. The PC12 cells are therefore an attractive model system not only for studies of neuronal differentiation but also for studies of neurofilament metabolism and disorders thereof.     

Transforming growth factor alpha, epidermal growth factor, and epidermal growth factor receptor expression in normal and diseased human adrenal cortex by immunohistochemistry and in situ hybridization

Because epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGFR) have been implicated in the regulation of adrenocortical function, we used immunohistochemistry and in situ hybridization of EGF and TGF-alpha to study 41 specimens of human adrenal cortex, including 10 normal specimens, 15 aldosteronomas, five Cushing's adenomas, six adrenocortical incidentalomas, and five carcinomas to determine what role these growth factors play in controlling human adrenocortical function. Neither immunoreactivity nor mRNA hybridization signals to EGF was detected in any specimens, and EGF therefore may exert its effects on adrenal function as an endocrine hormone. TGF-alpha expression was detected at both protein and mRNA levels in normal and neoplastic adrenal cortex, demonstrating that TGF-alpha is synthesized locally in human adrenal cortex. TGF-alpha expression was observed in the cells with increased steroidogenesis, including compact tumor cells and zona fasciculata cells with lipid depletion, but did not necessarily correlate with production sites of any specific steroid hormone. EGFR immunoreactivity was more widely distributed than TGF-alpha immunoreactivity. Both TGF-alpha and EGFR expression were markedly elevated in adrenocortical carcinomas. TGF-alpha and EGFR thus appear to be involved in biological function in both normal and neoplastic human adrenal cortex. In addition, TGF-alpha and EGFR may play important roles in some biological features of adrenocortical malignancy.     

Induction of epidermal growth factor (EGF) in the urogenital sinus by a brief treatment with androgens

To investigate whether estrogen receptor abnormalities are associated with resistance of breast cancer to endocrine therapy, we compared estrogen receptor mRNA between normal breast tissue and carcinoma. Using the RT-PCR, paired cancer and normal breast tissue specimens from 15 patients were analyzed. Exon-deleted variants were found in both tumor and normal tissue, with differences of variant expression between normal and tumor tissue being observed. One patient showed multiple deletions in the hormone-binding domain. Alterations in the amount and/or kind of variant ER expression may be important in determining the response of breast cancer to endocrine therapy.     

Inhibition of insulin receptor activation by insulin-like growth factor binding proteins

The insulin-like growth factors (IGFs) are transported by a family of high-affinity binding proteins (IGFBPs) that protect IGFs from degradation, limit their binding to IGF receptors, and modulate IGF actions. The six classical IGFBPs have been believed to have no affinity for insulin. We now demonstrate that IGFBP-7/mac25, a newly identified member of the IGFBP superfamily that binds IGFs specifically with low affinity is a high-affinity insulin binding protein. IGFBP-7 blocks insulin binding to the insulin receptor and thereby inhibiting the earliest steps in insulin action, such as autophosphorylation of the insulin receptor beta subunit and phosphorylation of IRS-1, indicating that IGFBP-7 is a functional insulin-binding protein. The affinity of other IGFBPs for insulin can be enhanced by modifications that disrupt disulfide bonds or remove the conserved COOH terminus. Like IGFBP-7, an NH2-terminal fragment of IGFBP-3 (IGFBP-3((1-87))), also binds insulin with high affinity and blocks insulin action. IGFBPs with enhanced affinity for insulin might contribute to the insulin resistance of pregnancy, type II diabetes mellitus, and other pathological conditions.     

c-fos gene expression in postnatal rat retinas with light/dark cycle

We examined the diurnal variation of c-fos gene expression during a 12:12 light/dark cycle in developing rat retinas by in situ hybridization histochemistry. c-fos Gene was not expressed before postnatal day 10 (P10) but was expressed on P15 in the outer nuclear layer throughout the dark period and in the inner nuclear layer and the ganglion cell layer during the light period. These results demonstrated that the earliest c-fos gene expression occurred between P11 and P15. The good correlation between the expression of c-fos gene and the genes coding for proteins involved in phototransduction, in terms of their diurnal variation and in their development, suggested that c-fos gene may play a role in the regulation of these genes in retinal cells during the light/dark cycle.     

Interaction of c-myc with transforming growth factor alpha and hepatocyte growth factor in hepatocarcinogenesis

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human TGF-alpha cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumorigenesis. Coexpression of c-myc and TGF-alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumors were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. These observations suggest that the interaction of c-myc and TGF-alpha, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion. These studies have now been extended to examine the interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in the transgenic mouse model. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Similarly, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine. Furthermore, we show for the first time that interaction of c-myc with HGF or TGF-alpha results in profoundly different outcomes of the neoplastic process in the liver.     

Overexpression of cellular Src in fibroblasts enhances endocytic internalization of epidermal growth factor receptor

Previous studies have demonstrated a requirement for the nonreceptor tyrosine kinase, cellular Src (c-Src), in epidermal growth factor (EGF)-induced mitogenesis and a synergistic interaction between c-Src and EGF receptor (EGFR) in tumorigenesis. Although endocytic internalization of EGFR may be thought to attenuate EGF-stimulated signaling, recent evidence suggests that signaling through Ras can be amplified by repeated encounters of endosome-localized, receptor. Shc.Grb2.Sos complexes with the plasma membrane, where Ras resides almost exclusively. Based on these reports, we examined EGFR trafficking behavior in a set of single and double c-Src/EGFR C3H10T1/2 overexpressors to determine if c-Src affects basal receptor half-life, ligand-induced internalization, and/or recycling. Our results show that overexpression of c-Src causes no change in EGFR half-life but does produce an increase in the internalization rate constant of EGF.EGFR complexes when the endocytic apparatus is not stoichiometrically saturated; this effect of c-Src on EGFR endocytosis is negligible at high receptor occupancy in cells overexpressing the receptor. In neither case are EGFR recycling rate constants affected by c-Src. These data indicate a functional role for c-Src in receptor internalization, which in turn could alter some aspects of EGFR signaling related to mitogenesis and tumorigenesis.     

Unliganded epidermal growth factor receptor dimerization induced by direct interaction of quinazolines with the ATP binding site

Receptor dimerization is critical for signaling by the epidermal growth factor receptor (EGFR) tyrosine kinase. This occurs after binding of the receptor's extracellular domain by ligand or bivalent antibodies. The role of other receptor domains in dimerization is less clear, and there are no examples of dimers induced by direct perturbation of the EGFR kinase domain. Submicromolar concentrations of AG-1478 and AG-1517, quinazolines specific for inhibition of the EGFR kinase, induced reversible receptor dimerization in vitro and in intact A431 cells. Consistent with the inhibitory effect of quinazolines on receptor kinase activity, the dimers formed lacked a detectable Tyr(P) signal. Quinazoline-induced EGFR dimerization was abrogated in vitro by ATP and the ATP analog adenyl-5'-yl imidodiphosphate. Receptors with a single-point mutation in the ATP binding site as well as wild-type EGFR with a covalent modification of the ATP site failed to dimerize in response to AG-1478 and AG-1517. These data suggest that EGFR dimerization can be induced by the interaction of quinazolines at the ATP site in the absence of receptor ligand binding. In SKBR-3 cells, the quinazolines induced the formation of inactive EGFR/ErbB-2 heterodimers, potentially sequestering ErbB-2 from interacting with other coreceptors of the ErbB family. Structural studies of the quinazoline interaction with the EGFR tyrosine kinase domain should allow for an analysis of receptor-specific chemical features required for binding to the ATP site and disruption of signaling, a strategy that can be perhaps applied to other tumor cell receptor systems.     

Prognostic value of epidermal growth factor receptor expression in endometrioid endometrial carcinoma

We report here a retrospective study of epidermal growth factor receptor (EGFR) expression in 140 patients with human endometrioid endometrial carcinoma (median period of follow-up, 43.8 months; ranging from 1 to 155 months). Tumor specimens were immunohistochemically examined for the overexpression of EGFR, and the correlation among EGFR status, various clinicopathologic parameters, and prognosis was statistically evaluated. Monoclonal antibody (clone 31 G 7), which recognizes the extracellular domain of the EGFR molecule, was used for immunostaining. Ninety-four of 140 cases were immunohistochemically positive for EGFR (67.1%). The presence or absence of EGFR did not correlate with surgical stage, depth of myometrial invasion (DI), or lymph node involvement, but did correlate with histological grade and patient's age. Furthermore, patients with EGFR-positive endometrial carcinoma had a statistically significant shorter length of survival than those with EGFR-negative tumors (P = .018). This trend is more apparent among the patients more than 50 years old (P = .003). When adjusted for surgical stage, DI, and patient age, EGFR status retained prognostic value by multivariate analysis. However, when adjusted for surgical stage, histological grade, DI, and patient age, EGFR status failed to retain prognostic value by multivariate analysis. The results of this study suggest that EGFR expression is correlated with histological grade and greater invasiveness of human endometrioid endometrial carcinoma.