Growth factor responsiveness and alterations in growth factor homeostasis in Syrian hamster embryo cells during in vitro transformation

Syrian hamster embryo (SHE) cells were investigated for their growth factor responsiveness as well as changes in growth factor homeostasis, including alterations in autocrine growth factor production and growth factor responsiveness, during in vitro transformation. For wild-type SHE cells, fetal bovine serum (FBS), epidermal growth factor (EGF) family members, platelet derived growth factor (PDGF) family members, fibroblast growth factor family members, interleukin-4, interleukin-9, oncostatin M, hepatocyte growth factor, erythropoietin and pituitary extract were found to be mitogenic. SHE cell mitogenesis was inhibited in response to transforming growth factor beta (TGF-beta) family members, interleukin-1 alpha, interleukin-1 beta and nerve growth factor. Additional experiments were conducted to study alterations in growth factor responsiveness to three SHE cell mitogens (FBS, EGF and PDGF) and one inhibitor of mitogenesis (TGF-beta) during SHE cell in vitro transformation. Alterations in either EGF, PDGF or TGF-beta responsiveness were observed in 7/8 SHE transformed lineages during the stepwise transformation process. Finally, 6/8 lineages underwent alterations which resulted in the production of autocrine growth factors during the transformation process. These results indicate that multiple alterations in growth factor homeostasis occur during the in vitro transformation process.     

Insulin-like growth factor I-dependent regulation of prolidase activity in cultured human skin fibroblasts

In metastatic breast cancer the goal to reach must be the best possible palliation with minimum discomfort for the patient. We reviewed our experience with radiotherapy (20 or 30 Gy), systemic therapy and brace. Among 2200 breast cancer patients, we extracted 28 potential candidates for resection. All of them developed new metastases outside the treated field within one year. Local control was achieved in 68%, and 80% of them had stable or better performance status at 3 months. From our analysis, even patients with a so called "solitary lesion" do not seem to have a better prognosis than others. We conclude that radiotherapy (with systemic therapy and a brace) is still first-choice treatment for vertebral metastases; CT-guided percutaneous biopsy can avoid worthless major operations. The role of surgery should be limited to neurological compression, severe mechanical instability and to salvage the failures of conservative treatment.     

Epidermal growth factor receptor-mediated motility in fibroblasts

Cell motility is induced by many growth factors acting through cognate receptors with intrinsic tyrosine kinase activity (RPTK). However, most of the links between receptor activation and the biophysical processes of cell motility remain undeciphered. We have focused on the mechanisms by which the EGF receptor (EGFR) actuates fibroblast cell motility in an attempt to define this integrated process in one system. Our working model is that divergent, but interconnected pathways lead to the biophysical processes necessary for cell motility: cytoskeleton reorganization, membrane extension, formation of new adhesions to substratum, cell contraction, and release of adhesions at the rear. We postulate that for any given growth factor some of the pathways/processes will be actively signaled and rate-limiting, while others will be permissive due to background low-level activation. Certain couplings have been defined, such as PLCgamma and actin modifying proteins being involved in cytoskeletal reorganization and lamellipod extension and MEK being implicated in detachment from substratum. Others are suggested by complementary investigations in integrin-mediated motility, including rac in membrane protrusion, rho in new adhesions, myosin II motors in contraction, and calpain in detachment, but have yet to be placed in growth factor-induced motility. Our model postulates that many biochemical pathways will be shared between chemokinetic and haptokinetic motility but that select pathways will be activated only during RPTK-enhanced motility.     

Respiratory activity and growth of human skin derma fibroblasts

Symptom control is the goal of palliative irradiation. Approximately 1 month is required before symptomatic relief is accomplished with radiotherapy. However, many patients with cancer-related pain do not receive adequate analgesics, and opioids are often not prescribed until patients fail to respond to palliative irradiation. The presenting symptoms of 108 patients who were referred to a multidisciplinary clinic for bone metastases were evaluated with the Wisconsin Brief Pain Inventory (BPI). This validated instrument evaluates the severity of pain using a 0-10 scale; 10 represents the worst pain imaginable. The population comprised 65 men (60%) and 43 women whose ages ranged from 33 years to 81 years; median age was 55 years, and 69% of patients were less than 65 years of age. Despite the presence of metastatic disease, 21% of patients were working full-time outside the home, and 6% were employed part-time outside the home; 13% were homemakers. Only 17 patients (16%) were unemployed. The time since diagnosis ranged from 2 weeks to 23 years; the median time since diagnosis was 22 months, and 30% of patients had been diagnosed with the past 6 months. Pain was a presenting symptom in 74% (N = 80) of patients at diagnosis. At its worst, the pain was rated as severe (levels 7-10) by 78% and intolerable (level 10) in 22% of the patients in the 24 hr prior to the clinic appointment. On average, the pain was rated moderate to severe (levels 4-10) in 79% and severe in 23% of patients. Only 45% of patients experienced good relief from the prescribed analgesics, and 23% of patients indicated that the prescribed analgesics were ineffective. This survey demonstrates that bone metastases incur significant pain that is often undertreated with analgesics before antineoplastic therapy is administered.     

Magnesium responsiveness to insulin and insulin-like growth factor I in erythrocytes from normotensive and hypertensive subjects

Depletion of intracellular free magnesium (Mg(i)) is a characteristic feature of insulin resistance in essential hypertension, but it is not clear to what extent low Mg(i) levels contribute to insulin resistance, result from it, or both. As insulin-like growth factor I (IGF-I) may improve insulin resistance, we investigated whether this peptide could similarly improve Mg(i) responsiveness to insulin in hypertension, and whether this effect was related to any direct IGF-I effect on Mg(i). 31P-Nuclear magnetic resonance spectroscopy was used to measure Mg(i) in erythrocytes from 13 fasting normotensive and 10 essential hypertensive subjects before and 30, 60, and 120 min after incubation with a physiologically maximal dose of insulin (200 microU/mL) and with different doses of recombinant human IGF-I (0.1-100 nmol/L). In normotensive subjects, IGF-I elevated Mg(i) (P < 0.05) in a dose- and time-dependent fashion, as did insulin (P < 0.05). However, in hypertensive subjects, maximal Mg(i) responses to insulin, but not to IGF-I, were blunted [insulin, 163+/-11 to 177+/-10 micromol/L (P=NS); IGF-I, 164+/-6 to 190+/-11.7 micromol/L (P < 0.05)]. Furthermore, for insulin, but not for IGF-I, cellular Mg(i) responsiveness was closely and directly related to basal Mg(i) levels (insulin: r=0.72; P < 0.01; IGF-I: r=0.18; P=NS). Lastly, blunted Mg(i) responses to insulin could be reversed by preincubation of hypertensive cells with IGF-I. We conclude that 1) both IGF-I and insulin stimulate erythrocyte Mg(i) levels; 2) cellular Mg(i) responses to insulin, but not to IGF-I, depend on basal Mg(i) levels, i.e. the higher the Mg(i) the greater the sensitivity to insulin; and 3) IGF-I potentiates insulin-induced stimulation of Mg(i) at doses that themselves do not raise Mg(i). These effects of IGF-I may underlie at least in part its ability to improve insulin sensitivity clinically. Together, these data support a role for IGF-I in cellular magnesium metabolism and emphasize the importance of magnesium as a determinant of insulin action.     

Oncogenic Ras modulates epidermal growth factor responsiveness in endometrial carcinomas

OBJECTIVE: To compare cryotherapy, laser vaporization, and loop electrical excision for treatment of squamous intraepithelial lesions (SILs). METHODS: Women at least 18 years old with biopsy-proven SIL, negative pregnancy tests, negative findings on endocervical curettage, satisfactory colposcopy examinations, and congruent Papanicolaou smear and biopsy results were assigned randomly to treatment after stratification by SIL grade, endocervical gland involvement, and lesion size; they were evaluated 1, 4, 8, 12, 16, 20, and 24 months after treatment. Data were analyzed using chi2 statistics, logistic regression analysis, and the Cox proportional hazards model. RESULTS: Of 498 patients assigned, 108 were excluded (most because of inadequate follow-up), leaving 390 (139 cryotherapy, 121 laser vaporization, 130 loop excision) for analysis. All were followed 6-37 months (mean 16). There were no statistically significant differences in complications, persistence (disease present less than 6 months after treatment), or recurrence (disease present more than 6 months after treatment). Risk of persistent disease was higher among women with large lesions (risk ratio [RR], 18.9; 95% confidence interval [CI], 3.2, 110.6). Recurrence risk was higher among women aged 30 years and older (RR, 2.1; 95% CI, 1.2, 4.3), those with human papillomavirus type 16 or 18 (RR, 2.1; 95% CI, 1.1, 4.0), and those who had had prior treatment (RR, 2.1; 95% CI, 1.1, 3.9). CONCLUSION: The data support a high success rate with all three modalities. No significant difference in success rates was observed between the three treatments in our population. Additional attention and research should be directed toward the higher risk patients identified above.     

Immunohistochemical localization of heparin-binding epidermal growth factor-like growth factor in normal skin and skin cancers

Heparin-binding epidermal growth factor (EGF)-like growth factor is a 22-kDa glycoprotein that was originally identified as a secreted product of cultured human macrophages. Although the growth factor mRNA has been identified in various cells and tissues, the tissue distribution of the protein itself has rarely been demonstrated. In this study, the EGF-like growth factor was detected immunohistochemically in a variety of human skin samples by indirect immunofluorescence using a polyclonal rabbit antiserum raised against residues 26-41 of mature heparin-binding EGF. The keratinocytes of a variety of epithelium-derived structures demonstrated reproducible, specific staining for the EGF. In normal tissues, this staining was prominent in the basal cells of the epidermis and in the epithelial cells lining epidermal appendages such as hair follicles, sebaceous sweat glands and eccrine sweat glands. In addition, specific staining was detected in skin cancers derived from the basal epithelial cell layer, including basal and squamous cell carcinomas of the skin, with no staining detected in melanoma specimens. Immunoreactive heparin-binding EGF was characteristically associated with the surface of cells. With minor exceptions, the immunoreactive sites are identical to the known EGF receptor distribution in the skin, and suggest that keratinocyte-derived heparin-binding EGF may act in concert with other EGF family members in processes such as skin morphogenesis and wound repair, as well as in the development of skin cancers.     

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

The dietary effect of 1,3-biseicosapentaenoyl-2-gamma-linolenoyl glycerol (STG) on the fatty acid composition of guinea pigs was examined and compared with that of an eicosapentaenoic acid ethyl ester (EPA-E) and of a soybean oil (SBO) diet. In terms of content of plasma lipid, EPA-E had a greater hypolipidemic effect than STG. On the other hand, in terms of EPA incorporation, contents of EPA in liver lipid were almost the same in the STG and EPA-E groups. Considering that the amount of EPA administered in the EPA-E group was almost 1.5 times that of the STG group, EPA may be absorbed more effectively as the glycerol ester than as the ethyl ester in guinea pigs. In all the tissue lipids, the STG group had a higher unsaturation index (UI) than the EPA-E group even though there is a lower UI in the STG diet than the EPA-E diet. These results suggest that greater amounts of desaturase products as a whole were synthesized in the STG group than in the other two groups. The dihomo-gamma-linolenic acid/arachidonic acid (DGLA/AA) ratio in plasma total lipids in the STG group was 3.5 times that of SBO group, and the DGLA/AA ratio in the EPA-E group was half that of the SBO group. In liver lipid, the ratios of DGLA/AA and EPA/AA in the STG group were 0.687 and 0.488 (phosphatidylcholine fraction) and 0.237 and 0.752 (phosphatidylethanolamine fraction), respectively. The ratio of DGLA/AA as well as the high EPA/AA ratio obtained in the present study with the STG diet may lead to physiological alterations, including enhanced synthesis of 1- and 3-series eicosanoids.     

Activation of the epidermal growth factor receptor by skin tumor promoters and in skin tumors from SENCAR mice

The present study was designed to further investigate the role of the epidermal growth factor receptor (EGFr) in mouse skin tumor promotion by evaluating the status of the EGFr in tumor promoter-treated mouse epidermis and in mouse skin tumors. Female SENCAR mice received three topical treatments of either the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or the nonphorbol esters okadaic acid and chrysarobin. Membrane proteins from SENCAR mouse epidermis were isolated 6 h after the last treatment, and the phosphotyrosine content of the EGFr and several potential substrates were examined by Western blot analysis. The results indicated that multiple applications of all three tumor promoters led to an increase in the phosphotyrosine content of the EGFr and also of several lower molecular weight proteins (M(r) approximately 80,000-85,000). Phosphorylation of PLC gamma 1 on tyrosine residues could not be detected in tumor promoter-treated mouse epidermis when the phosphotyrosine content of the EGFr was elevated or in cultured keratinocytes exposed to exogenous EGF. When two tyrosine kinase inhibitors (tyrphostins RG50864 and RG13022) were incorporated into the treatment regimens, the TPA-induced epidermal hyperplasia and cell proliferation were effectively blocked, and the TPA-stimulated EGFr tyrosine phosphorylation was significantly reduced. Examination of the phosphotyrosine content of epidermal membrane proteins isolated from skin papillomas revealed that the EGFr also had elevated phosphotyrosine levels. These results demonstrate that multiple topical treatments with both phorbol ester and nonphorbol ester tumor promoters lead to activation of the EGFr tyrosine kinase in mouse epidermis. In addition, these data suggest that signaling through the EGFr pathway plays an important role in the tumor promotion stage of multistage carcinogenesis in mouse skin.     

Human epidermal growth factor and the proliferation of human fibroblasts

The majority of the mRNA molecules in HeLa cells contain 1-2 residue(s) of m6Ap and one blocked, methylated 5' terminal "cap" structure. The hnRNA, which is longer than mRNA, contains both m6Ap and caps but 4-6 times as many m6Ap residues per chain. In addition, nuclear molecules contain T2 RNA ase-resistant, methyl-labeled oligonucleotides ("di-" and "tri-" nucleotides) which are not found in mRNA. Some of the dinucleotides may be precursors to the 2'-0-methylated nucleotides in the cap structures. These results are compatible with internal methylation of hnRNA molecules (both m6Ap and 2'-0-methyl) followed by hnRNA cleavage and the addition of the cap structure to generate at least some of the HeLa cell mRNA. It also appears that some hnRNA molecules, which are longer than most mRNA molecules, contain cap structures suggesting the derivation of some mRNA molecules from the 5' regions of hnRNA.     

Nerve growth factor in normal and psoriatic skin equivalent models

Our objective was to determine the pattern and time course of nerve growth factor expression in an established skin equivalent model that we have used in the past to study wound healing and psoriasis phenotypes. Skin equivalents were constructed in triplicate using normal neonatal foreskin keratinocytes plated on collagen gels containing fibroblast lines. These lines were derived from five specimens of psoriatic lesions, three specimens of normal skin from patients with psoriasis, and three specimens of eyelid skin from normal donors. Immunohistochemistry and a monoclonal nerve growth factor-b antibody were used to determine the pattern of protein staining over 2 weeks. We looked at the wound healing phenotype using the skin equivalent model for 7-14 days. When keratinocytes invaginate into the dermis of skin equivalents (beginning at around 7 days of growth), dark staining of nerve growth factor was seen under the basal membrane zone, suggesting that nerve growth factor serves in the development of the basal membrane zone and the epidermis, and may influence the migration of nerves through the basal membrane zone into the regenerated skin.     

All-trans-retinoic acid interacts synergistically with basic fibroblast growth factor and epidermal growth factor to stimulate the production of tissue inhibitor of metalloproteinases from fibroblasts

This report examines the effect of all-trans-retinoic acid in combination with basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) on collagenase and tissue inhibitor of metalloproteinases (TIMP) production from human foreskin and synovial fibroblasts. When 10(-5) M retinoic acid is applied in combination with 1, 10, and 100 ng/ml of either FGF or EGF to foreskin or synovial fibroblasts, this results in a dose-dependent synergistic increase in TIMP protein production which is greater than the additive effect of the agents by up to fourfold. These responses can be inhibited by the presence of specific neutralizing antibodies to bFGF and EGF, demonstrating that they result from the presence of the growth factors and not from an experimental artifact such as bacterial endotoxin. We have also found that retinoic acid potently inhibits bFGF- and EGF-stimulated collagenase protein production in both skin and synovial fibroblasts.     

Expression of growth factor mRNAs by human Tenon''s capsule fibroblasts

We determined the nucleotide sequences of Norwalk-like viruses in 10 PCR products from stool or oyster specimens obtained from four outbreaks of gastroenteritis in which shellfish was suspected as the cause in Shizuoka prefecture in Japan between 1987-94. The sequences were determined from nucleotide positions 4561-4852 (292 bp) in the polymerase region. Two types of sequences were detected. One (genotype 1) had 87% sequence homology with the prototype Norwalk virus, and the other (genotype 2) had 59% sequence homology. The sequences from isolates belonging to the same genotype were almost the same regardless of the year of isolation. Because sequences of 2 genotypes were detected in 2 of the 4 outbreaks, nested PCR was performed with genotype-specific primers to detect the presence of 2 genotypes in the same specimen. In 5 of 10 specimens, PCR bands were detected with both genotype-specific primers, indicating the coexistence of 2 genotypes in 1 specimen. We also detected two genotypes of Norwalk-like virus in an oyster from a sample implicated in one of the outbreaks which may provide direct evidence of oysters as the cause of the gastroenteritis.     

Decorin and glycosaminoglycan synthesis in skin fibroblasts from patients with systemic sclerosis

We investigated the expression of decorin and the synthesis of sulphated glycosaminoglycans (GAGs) in cultured fibroblasts from patients with early-stage systemic sclerosis (SSc). Decorin mRNA levels were 1.8-fold higher in SSc fibroblasts than in control fibroblasts. SSc fibroblasts also produced 2.3-fold more decorin core protein and 2.2-fold more sulphated GAGs including dermatan sulphate and chondroitin sulphate. Newly synthesized GAGs, in the presence of p-nitrophenyl beta-xylopyranoside, which elongates dermatan sulphate and chondroitin sulphate as an initiator, were increased tenfold and were mainly composed of dermatan sulphate and chondroitin sulphate. The rate of stimulation by the beta-xyloside was similar in SSc and control fibroblasts. These results suggest that the increased amount of dermatan/chondroitin sulphate in SSc fibroblasts reflects an enhanced expression of decorin core protein. Type I collagen mRNA levels in SSc fibroblasts were also increased together with its synthesis. Therefore, our results indicate that an altered decorin and collagen production may affect the organization of collagen fibres and the fibrotic process observed in patients with SSc.     

Epidermal growth factor enhancement of skin tumor induction in mice

Subcutaneous injection of epidermal growth factor 1. significantly shortened the latency period for the appearance of methylcholanthrene induced skin tumors and 2. increased the average number of papillomas elicited per mouse in both the Swiss Webster and C3HeB/FeJ strains.     

Synthetic chimeras of mouse growth factor-associated glandular kallikreins. II. Growth factor binding properties

Six chimeric constructs of the sequentially similar growth factor-associated kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--have been expressed, and their ability to generate complexes with epidermal growth factor (EGF) and beta-NGF, analogous to the high molecular weight forms (7S NGF and HMW-EGF) found in the mouse submaxillary gland, evaluated. The chimeras are distinguished by the interchange of three regions composing the amino, middle, and carboxyl terminal regions that encompass four surface loops possibly involved in specific growth factor interactions. Native beta-NGF (along with native alpha-NGF) formed complexes indistinguishable from naturally occurring 7S NGF, characterized by an alpha 2 beta gamma 2 structure (where beta-NGF is itself a dimer), with recombinant (r) gamma-NGF and with a chimera in which the amino terminal region from EGF-BP was substituted. Two other chimeras containing either the middle or carboxyl terminal regions of gamma-NGF showed weaker ability to form 7S complexes. Thus, all chimeras containing two segments from gamma-NGF retained at least some ability to form the 7S complex. rEGF-BP reacted weakly with EGF, but the chimera composed of the amino and middle segments of EGF-BP and the carboxyl terminal segment of gamma-NGF formed a nativelike HMW-EGF complex. None of the other chimeras appeared to bind EGF. These results identify amino acid positions within each kallikrein that participate in strong growth factor interactions and demonstrate that, outside of active site contacts, different regions of the kallikreins are involved in the binding of EGF and beta-NGF, respectively.     

In vitro study of gingival fibroblasts from normal and inflamed tissue: age-related responsiveness

Iodothyronine 5' deiodinase, which is mainly responsible for peripheral T3 production, has recently been demonstrated to be a selenium (Se)-containing enzyme. The structure of nuclear thyroid hormone receptors contains Zinc (Zn) ions, crucial for the functional properties of the protein. In the elderly, reduced peripheral conversion of T4 to T3 with a lower T3/T4 ratio and overt hypothyroidism are frequently observed. We measured serum Se and RBC GSH-Px (as indices of Se status), circulating and RBC Zinc (as indices of Zn status), thyroid hormones and TSH in 109 healthy euthyroid subjects (52 women, 57 men), carefully selected to avoid abnormally low thyroid hormone levels induced by acute or chronic diseases or calorie restriction. The subjects were subdivided into three age groups. To avoid under- or malnutrition conditions, dietary records were obtained for a sample of 24 subjects, randomly selected and representative of the whole population for age and sex. Low T3/T4 ratios and reduced Se and RBC GSH-Px activity were observed only in the older group. A highly significant linear correlation between the T3/T4 ratio and indices of Se status was observed in the older group of subjects (r = 0.54; p < 0.002, for Se; r = 0.50; p < 0.002, for RBC GSH-Px). Indices of Zn status did not correlate with thyroid hormones, but RBC Zn was decreased in older as compared with younger subjects. We concluded that reduced peripheral T4 conversion is related to impaired Se status in the elderly.