Epidermal Growth Factor Receptor Immunoreactivity Research Articles

Effect of tyrphostin on cell growth and tyrosine kinase activity of epidermal growth factor receptor in human gliomas

The effects of tyrphostin, a selective protein tyrosine kinase inhibitor, on epidermal growth factor (EGF)-stimulated cell growth and EGF-receptor tyrosine kinase activity were studied in four human glioma cell lines. Stimulation by EGF induced variable enhancements of cell growth as well as tyrosine phosphorylation of EGF receptor and intracellular target proteins in all glioma cell lines. The level of immunoreactive EGF receptor detected with antibodies against extra- and intracellular domains was moderate in all four glioma cell lines, but markedly decreased with the latter antibody in two glioma cell lines. This variation was associated with considerable reduction of the EGF-stimulated tyrosine autophosphorylation level. Tyrphostin inhibited dose-dependently the EGF-stimulated cell growth and tyrosine autophosphorylation in all glioma cell lines, and the optimum time for the maximum inhibitory effect on tyrosine autophosphorylation was 12 to 18 hours after treatment with tyrphostin. The antiproliferative activity of tyrphostin nearly correlated quantitatively with its potency as an inhibitor of the EGF-stimulated EGF receptor tyrosine kinase activity. Tyrphostin had no significant effect on the immunoreactive EGF receptor levels, on the affinity constants and numbers of EGF receptor, or on the down-regulation and specific internalization of EGF receptor in any glioma cell line, suggesting that the effects of tyrphostin are not likely to be the results of reduction in EGF receptor and EGF binding capacity. In addition, the serum-stimulated cell growth was also inhibited dose-dependently by higher concentrations of tyrphostin in all glioma cell lines. It might be suggested, therefore, that tyrphostin inhibits EGF-stimulated cell growth by a specific suppression of EGF receptor tyrosine kinase activity, and at higher concentrations there appears to be some degree of either nonspecific inhibition or inhibition of serum-stimulated protein tyrosine kinase activity to induce the cell growth inhibition of gliomas.

Expression of messenger ribonucleic acid and presence of immunoreactive proteins for epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and EGF/TGF alpha receptors and 125I-EGF binding sites in human fallopian tube.

Reverse transcription polymerase chain reaction (RT-PCR) revealed that the Fallopian tubes express epidermal growth factor (EGF), transforming growth factor (TGF alpha), and EGF receptor (EGF-R) mRNA. The RT-PCR product was verified by restriction enzyme digestion analysis. Immunohistochemically, EGF, TGF alpha, and EGF-R were localized in Fallopian tubes by use of specific antibodies to human EGF, mature fragments of human TGF alpha, and monoclonal antibodies to the extracellular binding domain of EGF-R. The tubal epithelial cells were the primary site of immunoreactive EGF, TGF alpha, and EGF-R, which were present to a lesser extent in the stromal cells, smooth muscle cell layers, fibroblasts of serosal tissue, and arterial endothelial and smooth muscle cells. Using antibodies generated against the amino and carboxy termini of TGF alpha precursor produced a similar cellular distribution to that observed for mature TGF alpha. The intensity of immunoreactive TGF alpha with these antibodies was similar to that seen with EGF. The ciliated and nonciliated epithelial cells in the ampullary and isthmus regions immunostained with similar intensity for EGF, TGF alpha, and EGF-R. The immunostaining for EGF, TGF alpha, and EGF-R was cycle-dependent, was considerably higher during late proliferative and early-to-mid-secretory phases than during early proliferative and late secretory phases of the menstrual cycle, and was reduced during the postmenopausal period. Specimens obtained 5-12 yr after tubal ligation immunostained for EGF, TGF alpha, and EGF-R similarly to sections from unligated tubes taken during the same phase of the cycle. Quantitative autoradiography of 125I-EGF binding generated a pattern similar to that of immunostaining for EGF-R binding. Net grain density/100 microns 2 calculated for different cell types indicated that the epithelial cells had a significantly higher grain density than did other tubal cell types (p < 0.05) without the cycle dependency seen in the immunohistochemical study. In summary, the results demonstrate that the human Fallopian tube expresses mRNA and contains immunoreactive proteins for EGF, TGF alpha, and EGF-R as well as binding sites for 125I-EGF. The cycle dependency and lower immunostaining in postmenopausal tubes suggest a potential regulation of their expression by ovarian steroids. The results imply the importance of EGF/TGF alpha in a variety of tubal biochemical and physiological functions and possibly early embryonic development.

Expression of EGFR, HER-2/neu, P53, and PCNA in Endometrioid, Serous Papillary, and Clear Cell Endometrial Adenocarcinomas

Expression of four biologic markers was studied in 69 cases of endometrial cancer to identify their association with cell type, decreased survival, and increased tumor metastasis. Cell types included endometrioid (n = 45), serous papillary (n = 16), and clear cell (n = 8). Immunohistochemical stains were employed to detect the presence of epidermal growth factor receptor (EGFR), HER-2/neu, p53, and proliferating cell nuclear antigen (PCNA). Analysis revealed that EGFR was expressed in 49%, HER-2/neu in 59%, p53 in 9%, and PCNA in 16% of tumor specimens. HER-2/neu overexpression was significantly associated with depth of myometrial invasion. p53 and PCNA immunoreactivity significantly correlated with nonendometrioid histology, although PCNA was less specific in labeling these less favorable cell types. EGFR immunoreactivity also significantly correlated with nonendometrioid cell types and tumor metastases at time of diagnosis. Seventy-seven percent of patients with metastatic disease were EGFR-positive versus 36% positivity in patients with no evidence of metastases (P < 0.002). For patients with endometrioid adenocarcinoma, evidence of EGFR overexpression decreased survival from 89 to 69% (P < 0.04). In the serous papillary and clear cell category, EGFR positivity decreased survival from 86 to 27% (P < 0.03). EGFR strongly correlates with tumor metastasis and patient survival in endometrial cancer. Altered expression of this oncoprotein may serve as a guide to prognosis and treatment in these patients.

Prognostic utility of epidermal growth factor receptor overexpression in endometrial adenocarcinoma

Overexpression of epidermal growth factor receptor (EGFR) has been reported in endometrial adenocarcinoma. A retrospective analytic study was designed to investigate its prognostic utility. Sixty-nine patients were studied with cell types that included endometrioid (n = 45), papillary serous (n = 16), and clear cell (n = 8). Patients' medical charts and survival data were reviewed. Assessment of EGFR overexpression was done at the protein level by the use of an anti-EGFR polyclonal antibody that reacts with the cytoplasmic membrane glycoprotein receptor in paraffin-embedded tissues. EGFR was overexpressed in 34 (49%) patients in whom immunoreactivity was limited to neoplastic cells. Initial bivariate analysis revealed significant correlations between EGFR immunoreactivity and histologic grade (r = 0.44, P < 0.001), metastasis (r = 0.38, P < 0.001), cell type (r = 0.30, P < 0.01), myometrial invasion (r = 0.30, P < 0.01), and patient age (r = 0.30, P < 0.01). Multiple logistic regression analyses showed that EGFR overexpression and nonendometrioid cell types are two independent statistically significant markers for the presence of metastases. EGFR immunoreactivity can significantly predict myometrial invasion, but after controlling for the histologic grade, its ability of significantly predict invasion was lost. EGFR overexpression was shown to be a statistically significant predictor of survival, even after controlling for patient age, histologic grade, and cell type. Expression of this oncoprotein may serve as an independent prognostic indicator and a guide to therapy in patients with endometrial cancer.

Distribution of Epidermal Growth Factor and Epidermal Growth Factor Receptor in Human Lung: Immunohistochemical and Immunoelectron-Microscopic Studies

The distribution of epidermal growth factor (EGF) and EGF receptor (EGFR) in adult human lung was examined by immunohistochemistry and immunoelectron microscopy. EGF immunoreactivity was observed in secretory granules and endoplasmic reticulum of serous acinar cells of bronchial glands. EGFR immunoreactivity was detected at the cell membrane of the basal cells and the bronchial surface nonciliated cells (indifferent cells) in the bronchus, the nonciliated bronchiolar cells (Clara cells) and the type II pneumocytes in peripheral lung. These results suggest that EGF is secreted into bronchial surface fluid from the bronchial glands and stimulates proliferation and/or differentiation of basal cells and indifferent cells of the bronchus, although the source of EGF responsive to Clara cells and the type II pneumocytes of peripheral lung was not determined.

Studies of c-jun oncogene expression in human breast using a new monoclonal antibody, NCL-DK4.

A new monoclonal antibody to human c-jun oncoprotein, designated NCL-DK4, has been produced. NCL-DK4 has been proved to be highly effective for use on formalin-fixed, paraffin-embedded tissues, enabling the study of c-jun expression at a cellular level in both normal and neoplastic human tissues. The expression of c-jun oncogene has been examined in normal, benign, and malignant breast tissues, and c-jun-specific immunoreactivity in carcinomas has been related to histological type, tumour grade, c-erbB-2, oestrogen receptor, progesterone receptor, and epidermal growth factor receptor expression. Normal and benign breast tissues showed c-jun-specific immunostaining, which was weaker and in fewer cells compared with the c-jun immunoreactivity observed in breast carcinomas. No relationship was found between the degree of immunostaining and the extent of proliferative changes in benign breast tissues. Ninety per cent of all breast carcinomas studied showed c-jun-specific nuclear staining. There were no statistically significant differences in the intensity of c-jun immunoreactivity among grade I, II, and III infiltrating ductal carcinomas. There was no significant relationship between c-jun oncoprotein expression and c-erbB-2, oestrogen, progesterone, and epidermal growth factor receptor immunoreactivity.

The prevalence of BCL-2 immunoreactivity in breast carcinomas and its clinicopathological correlates, with particular reference to oestrogen receptor status

BCL-2 protein plays a pivotal role in overriding programmed cell death (apoptosis), thus favouring a prolonged survival of normal and neoplastic cells. Expression of the bcl-2 gene has been documented in some human tumours (non-Hodgkin's lymphomas and prostatic adenocarcinomas), but findings in breast carcinomas have not been reported. We have used the monoclonal antibody 124 to investigate BCL-2 expression in 212 breast carcinomas, and to correlate it with the oestrogen (ER), progesterone (PR) and epidermal growth factor receptor (EGFR) status, and with other clinicopathological variables including tumour type, grade, stage, growth fraction (as evaluated by Ki-67 immunostaining), and p53 accumulation. Of the 212 carcinomas, 173 (81.6%) exhibited BCL-2 immunoreactivity in more than 25% of the neoplastic cells. BCL-2 immunoreactivity was strongly correlated with ER and PR expression (P < 0.00001), with the lobular type (P = 0.012) and with better differentiated neoplasms (P = 0.00003), whereas it was inversely correlated with EGFR (P < 0.00001), p53 (P = 0.0004) and Ki-67 (P = 0.0002) immunoreactivities. No association was found with tumour stage (T and N categories). We conclude that bcl-2 expression in breast cancers is related to the oestrogen-dependent transcription pathway.

Localization of immunoreactive epidermal growth factor receptor in neonatal and adult rat hippocampus

The regional and developmental expression of epidermal growth factor (EGF) receptor in rat hippocampus was investigated utilizing immunocytochemical techniques at the light and electron microscopic levels. EGF receptor immunoreactivity in adult hippocampus was compared to that found at postnatal day 7 (P7). While the receptor was observed in P7 hippocampus, immunostaining was more prominent in the adult hippocampus, especially in the pyramidal CA2 field. Ultrastructural analysis of this region revealed that the receptor was localized to the cell bodies of both P7 and adult neurons rather than the axons or dendrites. The expression of EGF receptor in selected regions of the adult brain was verified by Western blotting. These results demonstrate the presence of EGF receptor in rat hippocampus as early as P7, localize the receptor to the pyramidal cell body, and establish the hippocampal formation, particularly CA2, as a major site of EGF receptor expression in rat brain.

Epidermal growth factor receptor expression in demented elderly: localization to vascular endothelial cells of brain, pituitary and skin

We have previously demonstrated that expression of epidermal growth factor receptor (EGFR) was upregulated on vascular endothelial cells from brains of patients with dementia but not in brains from non-demented patients. The present study used a monoclonal antibody against EGFR to examine its expression in pituitary gland, scalp, abdominal skin and brain of 29 patients with and without various dementias and neurological deficits, as well as normal aged controls. Of 20 clinically demented patients examined postmortem, 15 exhibited EGFR immunoreactivity (IR) on brain and peripheral vascular endothelial cells. Examination of nine non-demented patients revealed only 1 patient with EGFR-IR. EGFR-IR was expressed on all blood vessels, regardless of size or location within the tissue examined. Ultrastructural analysis of EGFR revealed that in pituitary, like brain, the receptor was restricted to the lumenal cell surface of vascular endothelial cells. In all cases of EGFR-IR there was an absolute correlation between central nervous system and peripheral expression; if there was EGFR-IR in brain vessels it was present in skin. If there was no staining in brain there was no peripheral skin vessel staining; and vice versa. Increased EGFR expression may indicate proliferative or regenerative changes in the vasculature of affected patients and may provide a biological marker supporting the diagnosis of dementia by analysis of skin biopsy.

In situ evaluation of the relationship between epidermal-growth-factor-receptor status and tumor-cell proliferation in colon carcinomas.

The association between the expression of the epidermal-growth-factor receptor (EGFR) and tumor proliferation was studied in 69 resected human adenocarcinomas of the colon. EGFR was detected immunohistochemically using the monoclonal antibody (MAb) EGFRI. Tumor-cell proliferation was assessed with the MAb Ki-67 directed against a proliferation-associated nuclear antigen expressed only in proliferating cells. The percentage of Ki-67-positive tumor cells (Ki-67 index) was evaluated by the point-counting method. Forty-seven carcinomas contained detectable EGFR immunoreactivity. Statistical analysis failed to reveal correlations between the EGFR status and T, N and M stages or tumor differentiation. The mean Ki-67 index did not differ between EGFR-positive and -negative carcinomas. Seven tumors contained clearly distinguishable areas with different EGFR staining intensity. In these tumors with a locally heterogeneous EGFR expression, tumor proliferation also did not correlate with EGFR immunoreactivity. These in situ observations suggest that EGFR expression may not play an important role in the growth regulation of human colonic carcinomas.

Differential Immunoreactivity of Epidermal Growth Factor Receptor in Benign, Dysplastic and Malignant Prostatic Tissues

To investigate epidermal growth factor receptor (EGFr) presence in the prostate, monoclonal antibody (clone EGFR1) immunohistochemical examination of radical prostatectomy specimens was performed (n = 37). All prostatic specimens contained benign prostatic hyperplasia (BPH) and/or dysplasia (prostatic intraepithelial neoplasia or PIN), as well as prostatic carcinoma (CaP). Areas of dysplasia were further categorized as to the basal cell layer and the luminal cell area. BPH, PIN, and CaP tissues in each specimen were analyzed by a single observer and graded on a scale from 0–4+. Fifteen samples were also analyzed for EGFr content utilizing a Cell Analysis Systems (CAS 200) image cytometer.EGFr immunoreactivity of BPH basal cells was significantly higher than EGFr immunoreactivity in areas of CaP (p <0.001). EGFr staining of BPH basal cells was also significantly higher than that seen in PIN luminal cells (p <0.001). Immunoreactivity of EGFr in PIN basal cells was significantly higher than in PIN luminal cells (p <0.001). EGFr staining of basal cells in BPH tissues was higher than that seen in the PIN basal cell layer but the difference was not statistically significant (p = 0.06). The amount of staining present in PIN luminal cells was also significantly greater than in CaP tissues (p = 0.002).Quantitative image analysis utilizing the CAS 200 image cytometer was performed on BPH and CaP areas exclusively. EGFr immunoreactivity in basal cells of the BPH tissues was significantly greater than that seen in CaP tissues (p <0.001).The decreased EGFr immunoreactivity in CaP may reflect a differentiating role for EGFr in normal tissues. Loss of EGFr influence may be associated with an increased proliferative state in PIN and CaP. Destruction or alteration of the epidermal growth factor receptor by a protease, such as prostatic specific antigen, may also explain our findings. At the present time the meaning of the different amounts of EGFr in the various types of prostate tissues is unknown.

Epidermal growth factor receptor distribution in burn wounds. Implications for growth factor-mediated repair.

Epidermal growth factor (EGF) along with several related peptide growth factors has been shown both in vivo and in vitro to accelerate events associated with epidermal wound repair. EGF and transforming growth factor alpha act by binding to a common EGF receptor tyrosine kinase thereby initiating a series of events which ultimately regulate cell proliferation. This study examined the immunohistochemical localization of EGF receptor (EGF-R) in burn wound margins, adjacent proliferating epithelium, and closely associated sweat ducts, sebaceous glands, and hair follicles. Tissue specimens removed during surgical debridement were obtained from full and partial thickness burn wounds in 32 patients with total body surface area burns ranging from 2 to 88%. In the early postburn period (days 2-4), prominent staining for EGF-R was found in undifferentiated, marginal keratinocytes, adjacent proliferating, hypertrophic epithelium, and both marginal and nonmarginal hair follicles, sweat ducts, and sebaceous glands. During the late postburn period (days 5-16), EGF-R was depleted along leading epithelial margins; however, immunoreactive EGF-R remained intensely positive in the hypertrophic epithelium and all skin appendages. Increased detection of immunoreactive EGF-R and the presence of [125I]EGF binding in the hypertrophic epithelium correlated positively with proliferating cell nuclear antigen distributions. Thus, the presence of EGF-R in the appropriate keratinocyte populations suggests a functional role for this receptor during wound repair. Dynamic modulation in EGF receptor distribution during the temporal sequence of repair provides further evidence that an EGF/transforming growth factor alpha/EGF-R-mediated pathway is activated during human wound repair.

Epidermal growth factor receptor at endothelial cell and pericyte interdigitation in human granulation tissue

Angiogenic immature capillaries in human granulation tissue possess many cytoplasmic interdigitations between endothelial cells and pericytes (CIDEP). Epidermal growth factor (EGF) is a potent mitogenic polypeptide which accelerates angiogenesis in vivo and in vitro. We have recently demonstrated in immature capillaries that EGF is present at the CIDEP and proposed that the CIDEP may be involved in a signaling pathway for EGF (Wakui et al., 1990c. Microvasc. Res. 40, 285–291), This study follows that previous report. In the present study, I have investigated the ultrastructural localization of the EGF receptor (EGF-r) at CIDEP. Immunoreactivity for the EGF-r in immature capillaries was located strictly at the CIDEP in large numbers, at the coated pits and vesicles of endothelial cells, and at some lysosome-like structures of the endothelial cells and the pericytes. On the other hand, immunoreactivity for EGF-r was absent in mature capillaries. At the CIDEP, EGF-r immunoreactivity was present at the cell membrane and a few cytoplasmic elements at the tip of pericyte cytoplasmic projections, but it was completely absent at the corresponding endothelial membrane indentation. The present results support our proposed hypothesis that the CIDEP in immature capillaries act as a pathway for EGF transportation from the endothelial cell to the pericyte by a receptor-mediated process.

Quantitative Determination of EGF-R During Epidermal Wound Healing

Little is known about the intrinsic regulation of growth factors of cytokines during the normal epidermal wound-healing processes in skin. A simplified model of wounding (tape stripping to remove the stratum corneum) was used to study the role of epidermal growth factor receptors (EGF-R) in this process. Although the dynamics of EGF-R in epidermal wound healing have not been determined, the immunoreactive EGF-R that are present presumably play an active role. Prior studies show that 1) EGF-R are present in increased numbers in proliferative skin diseases; 2) a hypertrophic epidermis, closely resembling normal wound healing, is induced in mouse skin by EGF injections; and 3) exogenous topical EGF potentiates wound healing. The number of immunoreactive receptors as measured by an enzyme-linked immunosorbent assay (ELISA) and histologic methods increased prior to an increase in epidermal thickness, total protein, and DNA content. This early increase in the levels of EGF-R was followed by a sharp decline in EGF-R and subsequent decline in epidermal thickness (hypertrophy), total protein, and DNA levels. Alterations in the temporal sequence in these parameters indicate that the EGF-R-mediated signaling systems play an active role in epidermal wound repair.

Immunolocalization of epidermal growth factor receptor and c-erbB-2 oncogene product in human ovarian carcinoma.

Abnormalities of epidermal growth factor receptor (EGFR) and c-erbB-2 have been demonstrated to be correlated with aggressive biologic behavior in a variety of human cancers. To analyze the possible roles of these oncogenes in ovarian neoplasms, immunolocalization of EGFR and c-erbB-2 oncogene product was performed in 45 cases of human ovarian mucinous and serous cystadenomas, carcinomas of low malignant potential (LMP), and invasive carcinomas by employing antibodies against these oncogene products. EGFR immunoreactivity was present in 15 of 35 LMP and invasive carcinomas and 1 of 10 cystadenomas. On the contrary, immunoreactivity of p185, which is an oncogene product of c-erbB-2, was detected only in five cases of carcinoma and in no benign cystadenoma. These results indicate that EGFR may be involved in the neoplastic process in epithelial ovarian adenocarcinoma, especially mucinous carcinoma, but involvement of c-erbB-2 is probably not as prevalent as considered previously. Four of the five cases immunohistochemically positive for p185 were also positive for EGFR, which suggests that expression of EGFR and p185 is to some extent correlated in human ovarian carcinoma.

Epidermal growth factor receptor in synaptic fractions of the rat central nervous system.

Functional relationships between epidermal growth factor (EGF) and neural tissues have of late attracted increasing interest. However, in spite of reported EGF effects on neurons, the expression of the EGF receptor (EGF-R) has not yet been unambiguously demonstrated in these cells. This 170-kDa protein bears an intracellular tyrosine kinase domain in which activity is ligand-dependent. We give definitive evidence here for its presence in neonatal and adult rat neurons showing also, for the first time, its binding and functional tyrosine kinase activities in the synaptic region. Immunohistochemistry using a polyclonal antibody prepared against the receptor purified from rat liver showed positive staining localized exclusively to neurons without regionalization to any particular brain zone. Binding studies made in Percoll-obtained synaptosomes revealed specific high affinity 125I-EGF binding sites (Kd, 1.42 x 10(-10) +/- 0.58 M) accounting for 17% of total binding and a great majority of low affinity (Kd, 2.55 x 10(-9) +/- 0.35 M) binding sites. Higher binding capacity was found in synaptosomal fractions obtained from newborn rats. The identity of the synaptosomal EGF binding activity with the 170-kDA EGF-R protein was demonstrated by cross-linking experiments. Furthermore, EGF-Affi-Prep affinity chromatography adsorbs a 170-kDa protein with EGF-R immunoreactivity from whole homogenates of adult rat brain. Phosphorylation assays made in freeze-thawed or intact synaptosomes showed EGF-induced tyrosine phosphorylation in the range of 170-, 126-150-, 124-, 113-, 98-, and 70-kDa proteins including the EGF-R. Thus, the EGF-R/EGF regulatory system could have a role in synaptic function that remains to be explored.

Immunohistochemical localization of epidermal growth factor and its receptor during odontogenesis in the rat.

The expression of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFr) in developing teeth has been immunohistochemically studied in rat embryos (E-16 to E-21). Both EGF and EGFr showed a similar pattern of distribution. A very weak immunostaining was observed in the dental germ cells during the bud, cap, and bell teeth stages, as well as in few ectomesenchymal cells. In developed, but not erupted teeth, a moderate immunoreactivity for EGF and EGFr was present in the odontoblasts, in the ameloblasts and in the internal epithelial cells, but it was stronger in the dentine. In addition, the presence of EGF/EGFr was also observed in the intercalated ducts of salivary glands, primarily the submaxillary gland, in the maxillary bone cells, and in the cells of the peripheral and central nervous system. These results suggest that EGF has little or no effect during the early periods of tooth differentiation, whereas it is probably involved in the production of dentine. Moreover, EGF/EGFr seem to participate in the maturation and differentiation of other embryonic tissues such as tissues of the nervous system and bone.

The correlation of spiral arteries morphology with uterine artery doppler velocimetry

Objective.The goal of this work was to study the expression of epidermal growth factor receptor (EGFR) and c-erbB-3 and c-erbB-4 oncogenes in gestational trophoblastic diseases and normal first-trimester placenta.Study design. Paraffin sections of 16 cases of partial mole, 25 cases of complete mole, 10 cases of gestational choriocarcinoma, and 11 cases of therapeutic abortion were studied immunohistochemically for EGFR, c-erbB-3, and c-erbB-4 proteins. The presence of EGFR mRNA was studied using in situ hybridization.Results. Staining for EGFR was detected immunohistochemically in all cell types in gestational trophoblastic diseases and normal placenta. In situ hybridization for EGFR mRNA correlated with immunostaining for EGFR in all tissues studied. All 10 cases of choriocarcinoma exhibited strong immunoreactivity for EGFR. The levels of expression of EGFR in choriocarcinoma and syncytiotrophoblasts and cytotrophoblasts in complete mole were significantly greater than those in syncytiotrophoblasts and cytotrophoblasts in both normal placenta and partial mole (P < 0.01, P < 0.01). Expression of c-erbB-3 did not significantly differ among placental and gestational trophoblastic disease tissues and trophoblastic cell types except for significantly increased expression in choriocarcinoma as compared with cytotrophoblasts of partial mole (P = 0.02). The placenta, complete and partial mole, and choriocarcinoma tissues demonstrated similar immunoreactivity for c-erbB-4. Strong immunostaining for EGFR (P = 0.02) and c-erbB-3 (P < 0.01) in extravillous trophoblasts of complete mole was found to be significantly correlated with the development of persistent postmolar gestational trophoblastic tumor.Conclusion. The EGFR-related family of oncogenes may be important in the pathogenesis of gestational trophoblastic diseases. The increased expression of EGFR and c-erbB-3 in complete mole may also influence the development of persistent gestational trophoblastic tumor.

Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and EGF and PDGF beta-receptors in human endometrial tissue: localization and in vitro action.

Human endometrial tissue and primary stromal cell culture contain immunoreactive epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AB as well as EGF and PDGF-beta receptors. The immunostaining for EGF, EGF receptor, and PDGF beta-receptor were associated with endometrial luminal and glandular epithelial and stromal cells, whereas only the stromal cells contain immunoreactive PDGF-AB. The immunostaining intensity of EGF, EGF receptor, and PDGF-AB was similar in both phases of the menstrual cycle, whereas, PDGF-beta receptor immunostaining was highest in proliferative phase and considerably reduced, particularly in luminal and glandular epithelial cells in the secretory phase. In addition primary stromal cell cultures express EGF, PDGF-AB, and contain EGF and PDGF-beta receptors, and very low levels of PDGF-alpha receptor. 3H-Thymidine incorporation indicate that after 48 h of incubation in serum-free medium approximately 75-80% of stromal cells are quiescent. Incubation of quiescent stromal cells with 10% fetal bovine serum stimulate 3H-thymidine incorporation in a time-dependent manner reaching maximal after 30-48 h, with a doubling time of 38.2 h. EGF (1.5-15 ng/ml) stimulates 3H-thymidine incorporation by quiescent stromal cells (P less than 0.001). This effect was significantly reduced at concentrations above 15 ng/ml (P less than 0.005). PDGF-AB (3-10 ng/ml) and PDGF-BB (0.5-10 ng/ml) also stimulate 3H-thymidine incorporation in quiescent stromal cells compared to controls (P less than 0.005). The action of EGF (15 ng/ml) and PDGF-AB (10 ng/ml) was time dependent, reaching maximal after 36 and 48 h of incubation (P less than 0.002). Addition of PDGF-AB (10 ng/ml) to EGF (15 ng/ml) significantly enhanced the action of EGF or PDGF-AB used individually (P less than 0.001). 17 beta-estradiol or progesterone at 1 microM did not stimulate 3H-thymidine incorporation, although they were stimulatory in combination (P less than 0.001), they did not alter the action of EGF or PDGF when added in combination. These observations provide further evidence that human endometrial tissue contains specific immunoreactive EGF receptors. It also demonstrates the presence of immunoreactive EGF, PDGF-AB, and PDGF-beta receptors in endometrial tissue as well as stromal cells in primary culture. Both EGF and PDGF are mitogenic for endometrial stromal cells, suggesting an autocrine/paracrine role in modulation of endometrial cell growth and differentiation.

Epidermal growth factor stimulates cAMP accumulation in cultured rat cardiac myocytes.

We have previously shown that epidermal growth factor (EGF) augments cAMP accumulation in the heart and stimulates cardiac adenylyl cyclase via a G protein mediated mechanism (Nair et al., 1989). More recently, employing an antibody against the carboxy-terminus decapeptide of Gs alpha, we have demonstrated that Gs alpha mediates the effects of EGF on cardiac adenylyl cyclase (Nair et al., 1990). Since the heart comprises of a variety of cell types, the purpose of the studies presented here was to determine whether or not the effects of EGF on adenylyl cyclase were mediated in cardiac myocytes or noncardiomyocytes. Therefore, cultures of ventricular cardiomyocytes and noncardiomyocytes from neonatal rat hearts were established and characterized. Apart from the differences in cellular morphology, cardiomyocytes but not the noncardiomyocytes employed in our studies expressed the alpha- and beta-myosin heavy chain (MHC) mRNA and the beta-MHC protein. Additionally, as described previously, treatment of cardiomyocytes with thyroid hormone increased alpha-MHC mRNA and decreased the expression of beta-MHC mRNA, indicating that the cardiomyocytes employed in our studies were responding in a physiologically relevant manner. EGF in a time-dependent manner increased cAMP accumulation in the cardiomyocytes but not in noncardiomyocytes. Maximum and half-maximum effects were observed at 100 nM and 2 nM concentrations of EGF, respectively. As determined by the presence of immunoreactive EGF receptors and tyrosine phosphorylation of the 170 kDa protein in membranes of cardiomyocytes and noncardiomyocytes, both the cell populations contained functional EGF receptors. Therefore, the differential effects of EGF on cAMP accumulation in the two cell populations appear to be due to differential coupling of the EGF receptors to the adenylyl cyclase system rather than the absence of EGF receptors in noncardiomyocytes. Consistent with our previous findings in isolated membranes and perfused rat hearts, EGF-elicited increase in cAMP accumulation in cardiomyocytes did not involve activation of beta-adrenoreceptors and was abolished by prior treatment of cells with cholera toxin. Overall, our findings demonstrate that EGF-elicited increase in cAMP accumulation in the heart is the reflection of changes in cAMP content of cardiomyocytes and not noncardiomyocytes.