Epidermal Growth Factor Receptor mRNA Expression Research Articles

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness.

Adenovirus E1A down-regulates the EGF receptorvia repression of its promoter

The epidermal growth factor receptor (EGF-R), after activation by its ligands, stimulates a cascade of intracellular events leading to cellular proliferation. Its expression is increased in various forms of cancer as a consequence of altered regulation. Our objective was to study potential negative regulators of EGF-R expression; we investigated the effect of adenovirus E1A proteins. E1A proteins can exert both positive and negative effects on cell growth, depending on the cell type and cellular context, and have anti-tumorigenic features on human cancer cells. We show that human cell lines stably transformed with the adenovirus E1 region show significantly reduced expression of EGF-R protein and mRNA compared to their control, non-E1A-expressing counterparts. Furthermore, the promoter activity of EGF-R can be specifically repressed by E1A in transient co-transfection analysis in multiple cell types. Transfections with deleted promoter fragments and constructs containing short fragments of the putative E1A-responsive region fused to a heterologous promoter indicate that E1A-responsive elements are contained in a promoter region (from -150 to -76). Analysis of E1A mutants showed that both E1A gene products, 12S and 13S, repress EGF-R promoter activity and that full repression requires the presence of an intact CR1 domain.

Enhanced expression of EGF receptor and low frequency of ras mutations in X-ray-induced rat thyroid tumours.

Radiation is recognized as a carcinogenic factor for the thyroid gland. In this experimental study, oncogene expression was investigated in radiation-induced rat thyroid tumours. Forty 3-month-old Wistar rats received X-ray-irradiation to the neck region; 40 animals were untreated controls. After 14 months, thyroid tumours had developed in 25 of the 29 irradiated animals still alive; 76% of these tumours were considered malignant. No tumours developed in controls. Mutations of codons 12-13 and 59-63 of H-, K- and N-ras were analysed by PCR-SSCP (single-strand conformation polymorphism analysis) and sequencing of DNA from thyroid tissue. SSCP indicated a ras mutation frequency of 8%, but only one K-ras codon 12 (Gly-Cys) mutation was confirmed by sequencing. Protooncogene expression was analysed by mRNA slot blot hybridization analysis and immunohistochemistry. K-ras mRNA expression and EGF receptor mRNA and protein expression were significantly increased in the irradiated animals compared with controls, and in tumours versus nontumour tissue. This study of radiation-induced rat thyroid tumours demonstrates that ras expression may be subject to changes apart from activating mutations. Increased expression of EGF receptor in the tumours parallels the situation in human thyroid cancer.

EFFECTS OF AN ENVIRONMENTAL ENDOCRINE DISRUPTOR ON FETAL DEVELOPMENT, ESTROGEN RECEPTOR alpha AND EPIDERMAL GROWTH FACTOR RECEPTOR EXPRESSION IN THE PORCINE MALE GENITAL TRACT

Purpose We studied the effect of a potent reproductive tract toxin, 2,3,7,8-tetrachlorodibenzo-rho-dioxin, on fetal development and expression of estrogen receptor alpha and epidermal growth factor receptor (EGFR) in male swine. Materials and Methods Fetal domestic swine and miniswine were injected with 1 micro g./kg. dioxin on day 50 of gestation and removed near term (114 days). Germ cell counts were performed on sections of formalin fixed testes. Estrogen receptor alpha protein, and messenger ribonucleic acid (mRNA) and EGFR mRNA expression were analyzed in frozen tissue using Western blotting and semiquantitative reverse transcriptase polymerase chain reaction. Results Of 15 dioxin exposed male offspring 8 (53%) had genital anomalies, including cryptorchidism in 4, epididymal detachment in 1, epididymal atresia in 1 and vasal dilatation in 3, while 3 of 17 control male swine (18%) had incompletely descended testes (p = 0.06). High intra-abdominal testes were found in 3 of 4 cryptorchid dioxin exposed but no control male swine. Mean germ cell number per tubule was 4.0 +/− 1.1 and 2.7 +/− 0.7 in control and dioxin groups, respectively (p = 0.01). Estrogen receptor alpha protein and mRNA were identified in fetal uterus, testis, gubernaculum and epididymis. Protein levels were 2 to 3-fold higher in dioxin exposed testis, and mRNA levels were significantly lower in gubernaculum and epididymis. EGFR mRNA expression was similar in treated and control testis and epididymis. Conclusions Preliminary data suggest that dioxin produces cryptorchidism and wolffian duct anomalies in male swine exposed just before mid gestation. Germ cell counts and estrogen receptor alpha mRNA expression in gubernaculum and epididymis were significantly reduced, and estrogen receptor alpha protein expression in testis appeared to be increased by dioxin exposure. Aberrant regulation of estrogen receptor alpha expression by dioxin may contribute to reproductive tract anomalies in male fetuses.

Expression of HGF, KGF, EGF and Receptor Messenger RNAs Following Corneal Epithelial Wounding

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and their receptors have been associated with homeostasis and wound healing in the cornea. The purpose of this study was to examine the expression of the messenger RNAs for these growth factors and receptors in a wounded series of mouse corneas using in situ hybridization.In situ hybridization was performed with3H-labeled riboprobes on unwounded corneas and corneas at 30 minutes, 4, 12, 24, 48 and 72 hr, and 7 days after epithelial scrape wounds in Balb/C mice. Qualitative and semi-quantitative analyses were performed.Expression of HGF, KGF and EGF mRNAs in keratocytes in the unwounded cornea was low. EGF mRNA was also expressed in unwounded corneal epithelium. Following wounding, however, these growth factor mRNAs were markedly upregulated in keratocytes. EGF mRNA expression in the epithelium appeared unaffected by wounding. At seven days after wounding and several days following closure of the epithelial defect, HGF mRNA and KGF mRNA were still expressed at higher levels in keratocytes compared with unwounded corneas. No difference in expression of HGF or KGF mRNAs between limbal, peripheral corneal, or central corneal keratocytes was noted in the unwounded cornea, KGF receptor mRNA was prominently expressed throughout the unwounded corneal epithelium. HGF receptor mRNA and EGF receptor mRNAs were expressed at low levels in unwounded cornea epithelium. Following scrape injury, expression of HGF receptor mRNA and KGF receptor mRNA were markedly upregulated in the corneal epithelium, while no significant increase in EGF receptor mRNA expression was noted.These studies suggest a prominent role for HGF and KGF in modulating corneal epithelial wound healing following injury. Less prominent changes in EGF mRNA and EGF receptor mRNA in the corneal epithelium following wounding may suggest that EGF has more of a role in homeostasis in the mouse corneal epithelium.

Antisense oligonucleotides to the epidermal growth factor receptor.

Overexpression of the epidermal growth factor receptor (EGFR) has been observed in human breast tumors and is associated with poor prognosis in breast cancer patients. This would suggest that blocking the activity of the EGFR is a logical approach in the treatment of breast cancer. Three 20-mer phosphorothioate oligodeoxynucleotides were designed to target different regions of the human epidermal growth factor receptor (EGFR) mRNA. Several analogs of these oligodeoxynucleotides (the 2'-fluoro analog, the 2'-propoxy analog, and/or the 5-methyl cytosine analog) were also evaluated. We added these compounds to a human ovarian carcinoma cell line (SKOV3) and a human lung carcinoma line (A549), both of which overexpress the EGFR. All of these antisense oligonucleotides inhibited expression of the 10 kb EGFR mRNA (range: 22-97% inhibition) compared to a scrambled control oligonucleotide or an untreated control. Expression of the less prominent 5.6 kb EGFR mRNA band was also inhibited by all but two of the parent oligonucleotides. No inhibition of this 5.6 kb band was found with the control oligonucleotide. The reduction in the expression of EGFR mRNA by the three most potent antisense compounds was accompanied by a significant reduction of EGFR protein (90-98%) and in vitro growth inhibition of SKOV3 cells as compared to the control oligonucleotide.

Expression of transforming growth factor alpha and epidermal growth factor receptor in human bladder cancer

We analysed the expression of epidermal growth factor receptor (EGFr) and transforming growth factor alpha (TGF-alpha) in human bladder tumours. Tumour biopsies were obtained from 54 patients with primary bladder cancer (18 stage T1 and 36 stage T2-4). The protein and mRNA expression of EGFr and TGF-alpha were quantified by ELISA and competitive RT-PCR, respectively. The EGFr protein level was significantly increased in T2-4 tumours (0.44 x 10(-11); 0.0-27.5 x 10(-11) mol/g) compared with T1 tumours (0.0; 0.0-2.0 x 10(-11) mol/g) (median; range; 2p<0.01). The EGFr protein and mRNA level correlated (Spearman r=0.45, 2p<0.005, n=40). Co-expression of TGF-alpha protein and EGFr protein was significantly associated with muscle invasive tumours (T2-4) (chi-squared=7.9, df=3, p<0.05) and the TGF-alpha protein level correlated significantly with EGFr protein expression (Spearman r=0.56, 2p<0.0001, n=54). While tumour stage correlated with survival, no correlation was observed between survival and the expression of EGFr and/or TGF-alpha. In conclusion, human bladder tumours express both EGFr and TGF-alpha. The expression of EGFr and TGF-alpha are closely correlated, and the expression of EGFr and co-expression of EGFr and TGF-alpha correlate with tumour stage.

Decrease in the level and mRNA expression of LH-RH and EGF receptors after treatment with LH-RH antagonist cetrorelix in DU-145 prostate tumor xenografts in nude mice.

Using radioligand binding, RT-PCR, and Southern blot analyses, we evaluated whether agonist [D-Trp6]LH-RH and antagonist Cetrorelix could affect the levels of receptors for LH-RH and EGF and expression of mRNA for these receptors in DU-145 human androgen-independent prostate cancers xenografted into nude mice. Radioligand binding studies showed the presence of specific high affinity receptors for LH-RH and EGF in DU-145 prostate tumors. Cetrorelix, but not [D-Trp6]LH-RH significantly inhibited tumor growth. The concentration of LH-RH receptors was reduced by 22% (p<0. 05) and 67% (p<0.01) after 4 weeks of treatment with [D-Trp6]LH-RH and Cetrorelix respectively. The concentration of EGF receptors fell by 48% (p<0.05) in the [D-Trp6]LH-RH group, whereas Cetrorelix led to a 66% reduction (p<0.01). The expression of LH-RH and EGF receptor mRNA was investigated by RT-PCR analysis followed by Southern blotting. Densitometric analysis of the developed bands showed that the antagonist Cetrorelix decreased the expression of LH-RH receptor mRNA by 55% (p<0.01) compared to control group while the 20% reduction after treatment with the LH-RH agonist was non-significant. Treatment with [D-Trp6]LH-RH and Cetrorelix also reduced the expression of EGF receptor mRNA by 35% and 68% respectively (both, p<0.01) compared to control group. In conclusion, these data demonstrate that growth inhibition of DU-145 prostate tumors induced by prolonged administration of LH-RH antagonist Cetrorelix is accompanied by a marked decrease in the concentration of LH-RH and EGF receptors as well as in their mRNA levels.

Normalization of EGFR mRNA levels following restoration of wild-type p53 in a head and neck squamous cell carcinoma cell line.

Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by mutation of the p53 tumor suppressor gene and increased expression of the epidermal growth factor receptor (EGFR). To determine the potential link between p53 and EGFR expression, we examined the effect of overexpressing wild-type p53 on EGFR mRNA levels in HNSCC. In these studies, a temperature-sensitive (ts) p53 mutant was transfected into an HNSCC line which contained a deletion of one allele of the p53 gene and a mutation of the remaining allele. Following selection, six clones were isolated, characterized by Southern blot analysis, and stable expression of mutant p53 protein was confirmed by immunoblotting of clones grown at the mutant temperature. Total RNA was isolated from transfectants grown at both wild-type or mutant temperatures followed by Northern blot analysis to determine levels of EGFR mRNA expression at the two p53 conformations. Clones grown at the wild-type temperature (32.5 degreesC) demonstrated a 69% 13% decrease in EGFR mRNA compared with the same cells grown at the mutant temperature (39.5 degreesC; p=0.006) indicating that restoration of wild-type p53 reduces EGFR mRNA levels in this HNSCC cell line. These findings suggest that abnormalities in p53 may contribute to activation of EGFR gene transcription.

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 μg/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 proteint 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 opertion, the beneficial effect of EGF during adaptation is likely caused by other factors in addition to increased receptor expression.

Enhanced expression of heparin-binding EGF-like growth factor and its receptor in hypertrophied left ventricle of spontaneously hypertensive rats.

Although heparin-binding epidermal growth factor-like growth factor (HB-EGF) is thought to produce hypertrophy in isolated cardiomyocytes via an autocrine mechanism, the pathophysiological role of HB-EGF, in myocardial hypertrophy in vivo, is not yet known. To investigate the involvement of HB-EGF in cardiac remodeling associated with hypertension in vivo, we assayed the expression of HB-EGF mRNA and protein in the left ventricle (LV) during the development of left ventricular hypertrophy in spontaneously hypertensive rats (SHR). Prior to sacrifice and assay of HB-EGF and EGF-receptor (EGF-R) mRNA, morphologic and hemodynamic variables were measured in SHR and in age-matched Wistar Kyoto rats (WKY). At 5, 9 and 12 weeks of age, rats were killed, their hearts were removed, and the expression of HB-EGF and EGF-R mRNA and protein were measured. In addition, SHR and WKY were treated with enalapril, atenolol, or both for 4 weeks. In untreated SHR, double products (i.e. systolic blood pressure (sBP) multiplied by heart rate (HR), an index of mechanical load, peaked at 9 weeks. Expression of HB-EGF mRNA was also observed to peak in these animals at 9 weeks, while expression of EGF-R mRNA increased from 5 to 9 weeks, but remained constant thereafter. In untreated WKY, double products and EGF-R mRNA expression did not change over time, whereas the level of HB-EGF message increased gradually. Antibody to HB-EGF reacted primarily with myocyte membranes in SHR, whereas antibody to EGF-R reacted mainly with interstitial cells in these animals. The angiotensin-converting enzyme inhibitor, enalapril, markedly decreased sBP in SHR, whereas the beta 1-adrenoreceptor antagonist, atenolol, significantly decreased HR. While neither alone affected the expression of HB-EGF mRNA, their combination significantly reduced the expression of HB-EGF mRNA, as well as double products, in these rats, but had no effect on expression of EGF-R mRNA. The enhanced expression of HB-EGF mRNA and protein in LV of SHR suggest that this growth factor may play an important role during the early development of LV hypertrophy and cardiac fibrosis in SHR. The association between double products and HB-EGF expression suggest that the latter may be induced by increased mechanical load and may contribute, in turn, to cardiac remodeling.

Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expression.

Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene is frequently associated with malignant gliomas. One type of EGFR mutation in primary gliomas results in overexpression of an aberrant EGFR messenger RNA (mRNA) that lacks sequences of exons II through VI of the human EGFR gene. We observed that the aberrantly spliced EGFR mRNA contains a ribozyme cleavable sequence (5'-AAG GUA AUU-3') created by the joining of EGFR exon I to exon VII. We hypothesized that an appropriately designed ribozyme RNA could mediate site-specific cleavage of the aberrant EGFR mRNA and reduce the growth of aberrant EGFR-producing tumor cells. We synthesized aberrant EGFR mRNA substrates and a sequence-specific hammerhead ribozyme (abEGFR-rib) to examine the ribozyme's activity in vitro. We also constructed an abEGFR-rib plasmid and introduced it into ERM5-1 cells, which are murine NIH3T3 cells transfected to express an aberrant EGFR complementary DNA. We measured the growth potential of the cotransfected cells in culture and in nude mice. The synthesized abEGFR-rib efficiently and specifically cleaved aberrant EGFR mRNA substrates in vitro. Expression of the transfected abEGFR-rib suppressed expression of aberrant EGFR mRNA in ERM5-1 cells and reduced the growth of tumors formed by the cotransfected cells in nude mice. Finally, the incorporation of bromodeoxyuridine, a measure of mitotic activity, was also decreased in abEGFR-rib-producing ERM5-1 cells in vivo. Ribozymes targeted to aberrant EGFR mRNA can inhibit the growth of tumors formed by cells that express this mRNA.

The expressions of epidermal growth factor receptor mRNA and protein gene product 9.5 in developing rat brain

To clarify the role of epidermal growth factor receptor (EGFR) in brain development, especially with regard to neuron differentiation, EGFR mRNA expression was studied by in situ hybridization in embryonic day (E)12, E16, postnatal day (P)1, P4, P15, P29 and adult rat, using protein gene product (PGP) 9.5 as a neuron marker. The primary germinal zone (neuroepithelium) expressed neither PGP 9.5 immunoreactivity (IR) nor EGFR mRNA. In the developing brain, cells expressing EGFR mRNA but not PGP 9.5 IR were found in the secondary germinal zone such as the subventricular zone and cerebellar external germinal layer, the cortical plate and, in later stage animals, the fiber tracts. Cells expressing both EGFR mRNA and PGP 9.5 IR appeared in a differentiating field. In the adult brain, strong EGFR mRNA expression was observed in Purkinje cells, Golgi cells, some hippocampal cells and neurons of the diencephalon, pontine and medullary nuclei, and weak expression was seen in neurons of the cerebral cortex. These results suggest that EGFR is related to the process of differentiation and maturation of neurons and the maintenance of some types of adult neurons.

Developmental and hormonal regulation of transforming growth factor-alpha and epidermal growth factor receptor gene expression in isolated prostatic epithelial and stromal cells.

Androgen has an important role in development of the prostate, and the actions of androgen are mediated, in part, by locally produced growth factors. These growth factors are postulated to mediate stromal-epithelial interaction in the prostate to maintain normal tissue physiology. Transforming growth factor-alpha (TGF-alpha) is one of the growth factors that can stimulate prostatic growth. The expression of TGF-alpha is thought to be regulated by androgen. The expression of epidermal growth factor receptor (EGFR), which is the receptor of TGF-alpha and EGF, also may be regulated by androgen. The hormonal and developmental regulation of TGF-alpha and EGFR messenger RNA (mRNA) levels in isolated epithelial and stromal cells from rat ventral prostate was investigated. The expression of mRNA for TGF-alpha and EGFR was analyzed by a quantitative RT-PCR (QRT-PCR) procedure developed. Observations from this assay demonstrated that both epithelial and stromal cells expressed the mRNA for TGF-alpha and EGFR. TGF-alpha mRNA expression was constant during postnatal, pubertal, and adult development of the prostate. EGFR mRNA expression was elevated at the midpubertal period and decreased with age. After castration of 60-day-old adult rats, both TGF-alpha and EGFR mRNA were significantly enhanced. TGF-alpha mRNA expression was stimulated by EGF in stromal cells (4.5-fold increase) but was not changed by any treatment in epithelial cells. EGFR mRNA levels were stimulated by EGF and keratinocyte growth factor treatment and inhibited by testosterone treatment in epithelial cells. Stromal cell EGFR mRNA levels were not affected by any treatment. Both testosterone and EGF stimulated incorporation of 3H-thymidine into prostatic stromal and epithelial cells. Anti-TGF-alpha antibody significantly inhibited testosterone-stimulated 3H-thymidine incorporation into stromal cells and epithelial cells. Immunocytochemical localization of TGF-alpha and EGFR demonstrated expression on the luminal surface of epithelial cells within prostatic ducts, and minimal expression was observed in stromal cells. Results indicate that testosterone does not directly regulate TGF-alpha mRNA levels but does inhibit EGFR mRNA levels. Interestingly, anti TGF-alpha antibody suppressed the effect of testosterone on 3H-thymidine incorporation into prostatic stromal and epithelial cells. This finding suggests that testosterone may act indirectly on prostatic cells to influence TGF-alpha actions. TGF-alpha mRNA levels were influenced by EGF in stromal cells only, and EGFR mRNA levels were influenced by testosterone, EGF, and keratinocyte growth factor in epithelial cells. These observations suggest that regulation of TGF-alpha and EGFR is distinct between the cell types. In conclusion, a network of hormonally controlled growth factor-mediated stromal-epithelial interactions is needed to maintain prostate development and function.

Epidermal growth factor upregulates the expression of its own intestinal receptor after small bowel resection

Background/Purpose: Epidermal growth factor (EGF) binds to its enterocyte receptor and enhances intestinal adaptation after massive small bowel resection (SBR). To ascertain the mechanism for enhanced adaptation by EGF, we sought to determine the effect of EGF administration on in vivo expression of the intestinal EGF receptor after SBR. Methods : Male ICR mice underwent a 50% proximal SBR and then were assigned randomly to EGF (150 μg/kg/d) or saline by twice daily intraperitoneal injection. After 3 days, the ileum was harvested and total protein and DNA content were measured. Northern hybridization and a ribonuclease protection assay were used to detect qualitative and quantitative expression of EGF receptor mRNA. The remaining ileum was pooled for each group and Western blotting used to determine expression of EGF receptor protein. Results : EGF augmented adaptation after SBR as monitored by significant increases in ileal protein (2.7 ± 0.08 saline versus 3.9 ± 0.17 mg/cm EGF; P < .001) and DNA (55.8 ± 1.6 saline versus 104 ± 8.4 μm/cm EGF; P < .001) content. Northern blotting results showed a marked (>fivefold) increase in ileal EGF receptor mRNA, which was confirmed with the ribonuclease protection assay. Administration of EGF after SBR induced a similar expression of EGF receptor protein. Conclusions : EGF enhanced intestinal adaptation after SBR. This augmented response is associated with increased ileal expression of EGF receptor mRNA and protein. Increased EGF receptor expression and subsequent enhanced ligand/receptor activity may be one important mechanism for the beneficial effect of EGF administration during intestinal adaptation.

Effect of Ovariectomy on Mammary Gland Expression of TGF.ALPHA. and EGFR mRNAs and its Relation to Mammary Gland Involution in Mice.

To understand the relationship between transforming growth factor α (TGFα) and mammary gland involution, changes in the growth of normal and preneoplastic mammary glands, the expression of TGFα and epidermal growth factor receptor (EGFR) mRNAs in the mammary gland, and serum prolactin (PRL) level were examined in 3-month-old virgin SHN mice 4 to 108 days after ovariectomy (OVX). The formation of normal end-buds and preneoplastic hyperplastic alveolar nodules was significantly reduced after 9 days of OVX, associated with the decreased serum PRL level. Both TGFα and EGFR mRNA expression in the mammary glands declined 18 days after OVX. Thereafter, TGFα mRNA tended to increase, while EGFR mRNA continued to decrease. These observations suggest that the decline of both estrogen and PRL play major roles in the mammary gland involution after OVX, and that the decline of EGFR, through which TGFα manifests its effects, play a secondary role in this process.

Growth stimulatory effect of interleukin (IL)-1 alpha produced by oral squamous carcinoma cell lines

The expression of IL-1 alpha and its effect on the cell growth were examined in six human oral squamous carcinoma cell lines. All the cell lines expressed IL-1 alpha mRNA and protein at various levels. Particularly, HSC-2 and HSC-3 cells showed high level of the mRNA expression and secreted large amounts of IL-1 alpha into the culture fluid. Scatchard plot analysis of IL-1 alpha binding revealed that HSC-2 cells had high-and low-affinity receptors, whereas IL-1 alpha receptors on HSC-3 cells were of undetectable level. The cell growth of HSC-2 and HSC-3 cells was stimulated by IL-1 alpha and inhibited by anti-IL-1 alpha antibody or IL-1 receptor antagonist. The expression of IL-1 alpha mRNA by these cell lines was induced by either IL-1 alpha, epidermal growth factor (EGF) or transforming growth factor alpha (TGF-alpha). On the other hand, IL-1 alpha promoted the mRNA expression of TGF-alpha and EGF receptor. These findings indicate that IL-1 alpha acts as an autocrine growth stimulator for oral squamous carcinoma cells in vitro and its interaction with EGF/TGF-alpha/receptor system may play a role in this enhanced growth by IL-1 alpha.

Transforming growth factor-alpha mRNA and epidermal growth factor receptor mRNA expression in normal and neoplastic mammary glands of four strains of mice with different mammary tumor potentials.

Transforming growth factor-alpha (TGF alpha) and epidermal growth factor receptor (EGF-R) mRNAs were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) in the normal and neoplastic mammary glands of four strains of mice with different mammary tumor potentials (from highest to lowest potential): SHN, GR/A, SLN and C3H/He. At 2 months of age, when the mammary glands of these strains consisted mostly of normal tissue, the samples examined showed the positive expressions of both TGF alpha and EGF-R mRNAs in all strains (4-6 mice per group), except for EGF-R mRNA in the SLN mice, expressed in only 2 of 4 samples associated with no end-bud formation in the mammary glands. At 10 months, all of the samples from all four strains had a positive expression of TGF alpha mRNA. The EGF-R mRNA expression paralleled the degree of the formation of preneoplastic hyperplastic alveolar nodules (HAN) in all strains. These findings indicate that TGF alpha and EGF-R participate in the growth of the mammary glands, and that EGF-R especially contributes to the formation of end-buds at younger ages and to that of preneoplastic HAN at later ages. All of the samples of mammary tumors from four strains had positive expressions of both TGF alpha and EGF-R mRNAs.

&lt;italic&gt;De novo&lt;/italic&gt; expression of transforming growth factor-α in parathyroid gland tissue of patients with primary or secondary uraemic hyperparathyroidism

The factors involved in abnormal parathyroid cell secretory function and growth in patients with primary and secondary hyperparathyroidism are still incompletely understood. We studied the expression of transforming growth factor-α (TGF-α), epidermal growth factor (EGF) and EGF receptor (EGF-R) at the gene message and the protein level in parathyroid tissue obtained from six patients with primary hyperparathyroidism, 15 patients with secondary uraemic hyperparathyroidism and five subjects with normal parathyroid tissue, using in situ hybridization and/or immunostaining technique. We found a consistent expression of TGF-α mRNA and protein in parathyroid endocrine cells of all six cases of primary parathyroid adenoma and in nearly all cases of secondary hyperplasia, in contrast to the absence of expression in normal parathyroid tissue. A marked expression of EGF-R mRNA and protein was also found in four of five tissue samples of primary parathyroid adenoma, in 13 of 15 tissue samples of secondary parathyroid hyperplasia and in most samples of normal parathyroid gland tissue. EGF mRNA and protein expression was undetectable in the majority of parathyroid tissue samples examined. Since TGF-α is known to bind to the EGF-R, the finding of an increased expression of TGF-α at the gene message and the protein level, together with a strong expression of EGF-R, in hyperplastic and adenomatous parathyroid glands suggests that this growth factor interacts with its receptor to promote parathyroid cell proliferation, perhaps by an autocrine mechanism.

De novo expression of transforming growth factor-alpha in parathyroid gland tissue of patients with primary or secondary uraemic hyperparathyroidism.

The factors involved in abnormal parathyroid cell secretory function and growth in patients with primary and secondary hyperparathyroidism are still incompletely understood. We studied the expression of transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF) and EGF receptor (EGF-R) at the gene message and the protein level in parathyroid tissue obtained from six patients with primary hyperparathyroidism, 15 patients with secondary uraemic hyperparathyroidism and five subjects with normal parathyroid tissue, using in situ hybridization and/or immunostaining technique. We found a consistent expression of TGF-alpha mRNA and protein in parathyroid endocrine cells of all six cases of primary parathyroid adenoma and in nearly all cases of secondary hyperplasia, in contrast to the absence of expression in normal parathyroid tissue. A marked expression of EGF-R mRNA and protein was also found in four of five tissue samples of primary parathyroid adenoma, in 13 of 15 tissue samples of secondary parathyroid hyperplasia and in most samples of normal parathyroid gland tissue. EGF mRNA and protein expression was undetectable in the majority of parathyroid tissue samples examined. Since TGF-alpha is known to bind to the EGF-R, the finding of an increased expression of TGF-alpha at the gene message and the protein level, together with a strong expression of EGF-R, in hyperplastic and adenomatous parathyroid glands suggests that this growth factor interacts with its receptor to promote parathyroid cell proliferation, perhaps by an autocrine mechanism.