Proliferation of anterior cruciate ligament cells in vitro by photo-immobilized epidermal growth factor.

The purpose of this study was to explore the early treatment potential of anterior cruciate ligament (ACL) injuries using artificial juxtacrine stimulation by photo-immobilization of a growth factor. A photo-reactive epidermal growth factor complex (EGF-Az) was synthesized by conjugating EGF with N-(4-azidobenzoyloxy) succinimide followed by immobilization onto polystyrene culture plates using UV irradiation. ACL cells from human tissues (1 x 10(5)cells, 100 microl/well) were cultured as follows: control, no EGF; 50 microl native EGF; 50 microl EGF-Az immobilized; and 100 microl EGF-Az immobilized. The ACL cells were cultured long-term and evaluated for possible differences in their responses to EGF. An in vitro wound closure assay was developed to enable examination of cellular proliferation and migration. ACL cell proliferation was most evident in the photo-immobilized EGF culture group and was seen to increase in proportion to the amount of added EGF. In the in vitro wound closure assay, the lesioned area at 72 h after culture initiation was indistinguishable in the photo-immobilized cultures, but remained clearly visible in the controls. We conclude that photo-immobilized EGF induced rapid proliferation of ACL fibroblast cells by artificial juxtacrine stimulation and speculate that similar EGF immobilization onto bioabsorbable material (e.g., polyglycolic acid or polylactic acid) might contribute to a new therapy for the treatment of ACL injuries.

Effect of photo-immobilization of epidermal growth factor on the cellular behaviors.

We constructed photo-reactive epidermal growth factor (EGF) bearing p-azido phenylalanine at the C-terminal (HEGFP) by genetic engineering to investigate the possibility of immobilized EGF as a novel artificial extracellular matrix (ECM). The constructed recombinant protein was immobilized to glass surface by ultraviolet irradiation. A431 cells adhered both to HEGFP-immobilized and collagen-coated surfaces. Interaction between immobilized HEGFP and EGF receptors in the A431 cells was independent of Mg(2+) although integrin-mediated cell adhesion to natural ECMs is dependent on Mg(2+). Phosphorylation of EGF receptors in A431 cells was induced by immobilized HEGFP as same as soluble EGF. DNA uptake of hepatocytes decreased by immobilized HEGFP whereas it increased by soluble EGF. Liver-specific functions of hepatocytes were maintained for 3 days by immobilized HEGFP whereas they were not maintained by soluble EGF, indicating that immobilized HEGFP follows different signal transduction pathway from soluble EGF.

The role of ultraviolet irradiation and heparin-binding epidermal growth factor-like growth factor in the pathogenesis of pterygium.

Ultraviolet (UV) light is one of the major factors implicated in the pathogenesis of pterygium. The mechanism by which UV light induces this disease remains elusive. The aim of this study was to evaluate the effects of UVB irradiation on the expression of growth factors in cultured pterygium epithelial cells and to demonstrate their distribution within pterygium. We cultured pterygial epithelial cells from pterygium explants and these cells were exposed to 20 mJ/cm(2) of UVB. Total RNA was extracted at 0, 6, and 12 hours after irradiation. (32)P-labeled cDNA was synthesized and analyzed using microarray technology to determine the differential expression of 268 growth factor and cytokine related genes. Semiquantitative reverse transcriptase-polymerase chain reaction was used to corroborate this data. Conditioned media derived from cells exposed to UVB irradiation was analyzed for protein expression by enzyme-linked immunosorbent assay. Immunohistochemistry was used to evaluate the distribution of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in pterygium tissue. Analysis of the hybridization signals revealed that the genes encoding HB-EGF, fibroblast growth factor 3, and cytotoxic trail ligand receptor were consistently elevated at 6 and 12 hours after UVB treatment. HB-EGF mRNA was elevated 6.8-fold at 6 hours after irradiation and was augmented in culture supernatants after the same treatment. Furthermore, HB-EGF reactivity was identified in the epithelium and vasculature of pterygium by immunohistochemistry. HB-EGF was present in normal limbal epithelium, although it was not induced in cultured limbal epithelial cells by UV irradiation. HB-EGF is a potent mitogen, localized in pterygium tissue, and significantly induced by UVB in pterygium-derived epithelial cells. We postulate that this growth factor is a major driving force in the development of pterygia and a means by which UV irradiation causes the pathogenesis of pterygium.

Salivary epidermal growth factor levels decrease in patients receiving radiation therapy to the head and neck.

OBJECTIVE: The objective of this study was to assess changes in salivary epidermal growth factor (EGF) in patients receiving radiation therapy to the head and neck and to determine whether salivary EGF levels correlate with the severity of radiation-induced oral mucositis. STUDY DESIGN: Thirteen patients and 18 control subjects were enrolled in the study. Saliva was collected before, during (weekly), and after radiation therapy. Salivary total protein (TP) and EGF concentrations were measured and correlated with the severity of oral mucositis. The variability in normalized EGF (ngEGF/mgTP) values and mucositis scores were analyzed with analysis of covariance, and the adjusted correlation coefficient was calculated. RESULTS: EGF levels decreased (P =.004), whereas TP levels increased over time (P =.039). A strong correlation was seen with decreasing normalized EGF values and more severe mucositis (P =. 0001). CONCLUSION: A strong negative correlation between normalized EGF and mucositis severity suggests a possible role for EGF in the progression of radiation-induced mucosal breakdown.

Tumour-proliferative fraction and growth factor expression as markers of tumour response to radiotherapy in cancer of the uterine cervix.

This study seeks to define the role of pretreatment of evaluation of tumour growth fraction in cervical cancer and its relationship to the clinical course of the disease. In addition, it also seeks to explain whether cell kinetics and growth factor expression have an association with tumour response to radiotherapy and hence could be of value in the management of patients. All pre-treatment biopsies were analysed for the tumour-proliferative compartment by evaluation of Ki67 antigen expression and argyrophilic nucleolar organiser region (AgNOR) counts. Growth factor analysis was done by analysing for expression of epidermal growth factor (EGF), epidermal growth factor receptor (EGF-R) and transforming growth factors alpha and beta (TGFalpha, TGFbeta). A total of 152 patients were evaluated and a correlation obtained between pre-treatment status of the tumour-growth-fraction-associated markers and clinical outcome following radiotherapy. Such patients were either disease-free (group 1, n=106) or with residual/recurrent disease (group 2, n=46) at a 16-month follow-up. Pre-treatment analysis of AgNOR significantly correlated to disease status after treatment (r=-0.517, P=0.0000). This may be due to an effect of cell proliferation. Lower AgNOR counts were significantly associated with recurrent/residual tumours, suggesting that increased proliferative activity may be a positive prognostic indicator. Similar results were also obtained for the other proliferation-associated marker Ki67 (r=-0.443, P=0.0000). Expression of EGF and EGF-R also showed significant pre-treatment correlations with the final disease outcome (r=0.248, P=0.031 and r=0.503, P=0.0000 respectively). Both these markers were expressed more by patients belonging to group 2. The opposite was the case for TGFalpha, where patients belonging to group 1 showed higher values (r=0.417, P=0.0001). The other growth factor investigated, TGFbeta, also showed a conspicuous differential expression in the two groups of patients (r=-0.604, P=0.0000). Group 1 patients showed mostly mild to moderate expression while most group 2 patients were negative for the growth factor. It therefore appears that tumours with high AgNOR counts and Ki67 index, along with expression of the two types of transforming growth factor (alpha and beta), responded better to radiotherapy.

Photo-immobilization of epidermal growth factor enhances its mitogenic effect by artificial juxtacrine signaling.

Photo-reactive epidermal growth factor (EGF) was synthesized by coupling EGF with azidobenzoic acid and was immobilized onto the wells of a polystyrene culture plate by photo-irradiation. The photo-immobilized EGF enhanced the growth of anchorage-dependent cells more than native or azidobenzoyl derivatized EGF. A small amount of photo-immobilized EGF was sufficient to enhance the growth of cells and the maximal mitogenic effect was greater than that of native or derivatized EGF. On the other hand, the photo-immobilized EGF did not enhance growth of anchorage-independent cells. In addition, signal transduction in the cells adhered only on the EGF-immobilized surface was observed by staining of phosphotyrosine residues by anti-phosphotyrosine antibodies. These results showed that the enhanced cell growth was due to direct interaction between the cells and the immobilized EGF. Photo-immobilization could be a universal means of fixing growth factors onto an artificial matrix that is devoid of chemically functional groups scaffolding growth factors and could provide a new tool to elucidate signal transduction mechanism and could lead to the development of a new protein-free cell culture system or tissue engineering materials.

Immunohistochemical and quantitative changes in salivary EGF, amylase and haptocorrin following radiotherapy for oral cancer.

Epidermal growth factor (EGF), amylase and haptocorrin are molecules produced in the salivary glands. The aim of the present study was to determine immunohistochemical and quantitative alterations in EGF as compared with haptocorrin and amylase following radiotherapy for oral cancer. Changes in the salivary secretion of EGF are of interest because of the importance of EGF in mucosal regeneration. Immunohistochemical studies on normal tissue from parotid and submandibular glands have demonstrated EGF in the serous acini with a tendency to single cell expression in the parotid gland. Amylase has been found in the serous acini of both the submandibular and parotid glands. Haptocorrin was localized in the duct system of both glands. In the submandibular glands with radiotherapy induced sialoadenitis only very few acini with weak or no staining for EGF and amylase were demonstrated, while no changes were observed in the staining for haptocorrin. Analysis on stimulated whole saliva samples collected from 20 healthy individuals and from 20 patients prior to, and 1, 2 and 3 weeks following radiotherapy showed significant reduction in salivary contents of EGF and amylase after treatment as expressed per g protein (p < 0.0002). The salivary content of haptocorrin increased significantly after treatment (p < 0.002). These alterations may be explained by the different cellular sites of the molecules studied, the serous acini being more sensitive to ionising radiation than the duct system. The concentration of EGF in saliva before treatment was significantly higher in patients than in the control group (p < 0.02), which may indicate that the tumors induce increased secretion of salivary EGF, or alternatively that the oral tumors contribute with EGF to the saliva. In conclusion we have demonstrated a reduction in the mitogenic peptide EGF both immunohistochemically and quantitatively following irradiation for oral cancer, results which may contribute to the understanding of the clinical signs of mucositis.

Inhibition of epidermal growth factor binding system by ionizing radiation in A431 human squamous carcinoma cells.

To elucidate the effect of ionizing radiation on the membrane anchored signal transduction, the binding of 125I epidermal growth factor (EGF) to its receptor (EGF-R) and the EGF-dependent EGF-R tyrosine phosphorylation were examined in a human squamous cell carcinoma cell line, A431. The significant suppression of 125I EGF binding to A431 cells was observed from 3-5 h after 10 Gy irradiation, whereas this inhibition was not observed both in non-irradiated and in 5 Gy-irradiated cells. This phenomenon was mediated by the protein kinase C pathway, because the inhibition was not observed in cells which had been pretreated with phorbol ester and treated with an inhibitor of the enzyme, H7. Scatchard analysis showed that the receptor affinity was decreased. In contrast, the level of EGF-dependent EGF-R-tyrosine phosphorylation was not decreased, compared with non-irradiated cells. These results suggest that ionizing radiation may modulate the function of EGF/EGF-R interaction through the direct activation of protein kinase C.

Ultraviolet-B (290-320 nm)-irradiation inhibits epidermal growth-factor binding to mammalian cells.

Mitogens, such as polypeptide growth factors and phorbol ester tumor promoters, act by binding to specific receptors and inducing a pleiotropic response in cultured mammalian cells, which results in the induction of cellular proliferation. An early effect of such agents is the inhibition of binding of epidermal growth factor (EGF) to its receptor. Ultraviolet radiation has also been shown to induce a proliferative response in vivo and in vitro and to act as a tumor promoter in animal skin. We, therefore, examined the effect of ultraviolet radiation (UVB - 290-320 nm) on EGF binding to cells in culture. We found that UVB (100-300 J/m2) induced a rapid, dose-dependent inhibition of EGF binding in a mouse fibroblast cell line, which resulted from a decrease in both number and affinity of binding sites. Phosphorylation of the EGF receptor by protein kinase C (PKC) is not likely to be the mechanism for inhibition, since UVB treatment did not result in PKC activation or modulation of phorbol diester binding.

Growth regulation in X-irradiated mouse skin; the possible role of chalones.

Extracts of hairless mouse skin were tested for their content of epidermal G1 inhibitor and G2 inhibitor at daily intervals after X-irradiation with 4 500 or 2 250 rad. After either dose the skin extracts lacked G1 inhibitory activity on days 5 and 6 respectively after irradiation. This coincided with the time when the epidermal mitotic rate again became normal and started a period of over-shoot. The time interval of 5-6 days corresponds to the turnover time of the differentiating cells in hairless mouse back epidermis. The findings indicate that the proliferating cells in epidermis can respond to changes in local chalone concentration, even after X-irradiation at the tested doses, and that the irradiated epidermal cell population still retains some important properties inherent in a cybernetically regulated system. The local G2-inhibitory activity also varied after irradiation, but these variations could not be directly related to the corresponding mitotic rates.