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Biomedical subjects

R M Lyall

Publications and source records attributed to R M Lyall.

16 recordsLinked to original sources
PubMed1991

The antiproliferative effects of tyrosine kinase inhibitors tyrphostins on a human squamous cell carcinoma in vitro and in nude mice.

Many human tumors of epithelial origin contain cells overexpressing the epidermal growth factor (EGF) receptor, and there is convincing evidence that cancer cell growth is correlated with the loss of the normal regulation of the EGF receptor signal transduction pathway. Some cancers are clearly dependent on activation of the EGF receptor for their proliferation. Recently, a class of compounds, tyrphostins, which inhibit the protein tyrosine kinase activity of the growth factor receptor, have been described. In this report, we have examined the antiproliferative effects of potent new tyrphostins on a well-characterized human squamous cell carcinoma in vitro and in vivo. We found that two of these compounds (RG-13022 and RG-14620) suppressed not only EGF-stimulated cancer cell proliferation in vitro but also tumor growth in nude mice. RG-13022 also increased the life span of these tumor-bearing nude mice. When administered to tumor-bearing nude mice together with monoclonal antibodies to the EGF receptor at a suboptimal dose which had no effect alone, inhibition of tumor growth was markedly enhanced. These data suggest that tyrphostins have potential as anticancer agents.

Animals↗
PubMed1989

Tyrphostins inhibit epidermal growth factor (EGF)-receptor tyrosine kinase activity in living cells and EGF-stimulated cell proliferation.

Synthetic compounds called tyrphostins were examined for their effects on cells which are mitogenically responsive to epidermal growth factor (EGF). We studied in detail the effects of two tyrphostins on EGF binding, tyrosine phosphorylation in intact cells, EGF-receptor internalization, and mitogenesis. These compounds inhibited EGF-stimulated [3H]thymidine incorporation in a specific manner and the degree of selectivity varied. Both compounds inhibited EGF-stimulated receptor autophosphorylation and tyrosine phosphorylation of endogenous substrates in intact cells at doses that correlated with the IC50 for [3H] thymidine incorporation. These results are consistent with the notion that tyrosine phosphorylation is a crucial signal in transduction of the mitogenic message delivered by EGF. The compound RG50864 demonstrated specificity at inhibiting EGF-stimulated cell growth compared with stimulation with either platelet-derived growth factor or serum. For both compounds RG50864 and RG50810, long term exposure (16 h) of cells to tyrphostins was required for optimal inhibition because of the instability and slow action of these compounds. Tyrphostins did not alter cell surface display of EGF-receptor, EGF binding or EGF-induced internalization, degradation, and down-regulation of EGF receptors. These novel synthetic inhibitors, specific for EGF-receptor kinase, offer a new method to inhibit EGF-stimulated cell proliferation which may be useful in treating specific pathological conditions involving cellular proliferation, including different types of cancers.

Animals↗
PubMed1988

Expression of acidic fibroblast growth factor cDNA confers growth advantage and tumorigenesis to Swiss 3T3 cells.

Acidic fibroblast growth factor (aFGF), a polypeptide with a mol. wt of approximately 16,000, is a potent mitogen for a variety of cells and shares 55% amino acid sequence identity with basic FGF. The recent isolation of three new oncogenes which share 35-45% amino acid sequence similarity with the FGFs suggests that the coding sequences for the FGFs themselves may be oncogenic under certain circumstances. To test this hypothesis, we cotransfected 3T3 NR6 cells with factors expressing the aFGF coding sequence and the bacterial neomycin gene. The aFGF produced by cotransfected cells was found only in the cellular homogenate and not in medium conditioned by the cells. Cells expressing aFGF grew to 10 times the density of control cells at saturation and were multilayered and disorganized, similar to transformed cells. The cotransfected cells do not grow in soft agar, but show enhanced soft agar growth relative to controls in the presence of added aFGF and heparin. The aFGF-producing cells formed small, non-progressive tumors when injected subcutaneously into nude mice. Our data suggest that expression of aFGF in NR6 cells results in enhanced growth, and that several traits characteristic of the transformed phenotype are partially expressed.

Animals↗
PubMed1987

Isolation of human KB cell lines resistant to epidermal growth factor-Pseudomonas exotoxin conjugates.

Mutants of the human KB carcinoma cell line resistant to a cytotoxic conjugate of epidermal growth factor (EGF) and Pseudomonas exotoxin (PE) were selected. EGF-PE and the drug verapamil, which enhanced EGF-PE cytotoxicity, were used in the selection process. These mutants also showed some cross-resistance to PE. All of the EGF-PE resistant variants displayed lower levels of 125I-EGF binding, 20-50% of parental KB levels, without altered affinity for EGF and grew at a slower rate than the parental cell line KB-3-1. These results indicate that EGF-PE resistant KB cells have a complex phenotype which includes a reduction in the number of EGF receptors and reduced sensitivity to unconjugated PE. Resistance to toxin-conjugates, although pleiotropic, is specific and does not lead to resistance to multiple other anticancer drugs, nor are independently selected multidrug resistant KB lines resistant to PE. These results argue that protocols for cancer treatment could effectively use specifically designed cytotoxic toxin conjugates as an adjunct to conventional chemotherapy.

Bacterial Toxins↗
PubMed1987

An angiogenic growth factor is expressed in human glioma cells.

Progression to increased malignancy frequently occurs in human brain tumors of glial origin and usually involves neovascularization--a massive proliferation of endothelial cells into the tumor tissue. We have shown previously that subversion of a normal growth factor-related pathway is frequently associated with human gliomas. Here we show that human glioma cell lines express the gene encoding the angiogenic peptide endothelial cell growth factor (ECGF) or acidic fibroblast growth factor (a-FGF) and that an ECGF-like polypeptide is produced by these cells. The glioma-derived growth factor was partially purified from cell extracts by heparin-Sepharose affinity chromatography where it eluted at 1.5 M sodium chloride. On reversed-phase h.p.l.c., growth factor activity for endothelial cells was eluted at the same concentration of acetonitrile as found for bovine brain-ECGF, also a potent mitogen for endothelial cells. Moreover, human glioma cells possess specific cell surface receptors for ECGF and are mitogenically stimulated by exogenous addition of this growth factor. Glioma derived-ECGF may therefore have a dual influence: first, by autocrine growth-stimulation of human gliomas and, second, by paracrine-stimulation of endothelial cell proliferation which results in neovascularization of the tumor tissue.

Cell Line↗
PubMed1987

A biochemical approach to periodontal regeneration. AFSCM: assays for specific cell migration.

Recent investigations on regeneration of the periodontium have attempted to define factors involved in the formation of a new connective tissue attachment. One essential biological event involved in tissue regeneration is directed cell migration (chemotaxis). Extracellular matrix proteins have been shown to influence chemotaxis, cell proliferation and differentiation. Recently, the extracellular matrix proteins, fibronectin (FN) and laminin (LM), and the polypeptide, endothelial cell growth factor (ECGF), have been shown to stimulate a variety of biological processes. Current assay systems which attempt to define cell migration are the Boyden chamber assay and a random cell migration assay. Neither assay system adequately defines in vivo cell migration. Here we present a new in vitro assay system that tests the capacity of several biological response modifiers applied on dentin to stimulate a chemotactic and proliferative response from various cell types. The assay system consists of two types of assays. Assay I measures the chemotactic activity of test substances bound to dentin. In this assay cells must actively move through a filter (Nuclepore) towards a factor bound to dentin. Assay II examines the ability of dentin-bound biological response modifiers to stimulate directed movement and proliferation of cells on dentin surfaces. We report that periodontal ligament (PDL) cells migrate towards FN and ECGF; that PDL cell migration is enhanced when dentin is preconditioned with tetracycline HCl; that PDL cells have an increased proliferative response when dentin is conditioned with both FN and ECGF; that gingival epithelial cells have increased migratory and proliferative responses when LM is used to condition dentin; and that there is a reciprocal utilization of biological response modifiers by gingival epithelial cells and PDL cells.

Cell Division↗
PubMed1986

Tumor cell metastasis.

Local tissue invasion and the formation of metastatic lesions are characteristic properties of many malignant tumors. The formation of metastases is a complex process involving the passage of tumor cells from the site of the primary bulk tumor through successive connective tissue barriers, ultimately resulting in the growth of secondary tumor cell colonies in distinct target organ locations. At many stages in the metastatic process, tumor cells interact with multiple components of the extracellular matrix. Recently, the importance of basement membrane as a barrier to invasive cells has been recognized. In the course of the transition from in situ to invasive carcinoma, normal or dysplastic epithelial cells residing on a basement membrane are replaced by neoplastic cells which subsequently invade the basement membrane and enter the underlying stroma. Once in the stroma, tumor cells can then penetrate the walls of blood vessels or the lymphatic system and enter into the circulation. Circulating tumor cells next arrest in the lumina of small vessels, invade the vessel wall, and leave the circulation. These cells are now directly exposed to the extracellular matrix of a target organ where they may grow to form secondary tumors. Throughout the metastatic process tumor cells are thus in contact with, and are potentially responsive to, various components of the extracellular matrix. This review provides a survey of the recent advances in our understanding of the interactions of metastatic tumor cells with the extracellular matrix. Specifically, the role of basement membrane as a barrier to metastatic tumor cells is examined.

Animals↗
PubMed1986

Laminin promotes rabbit neutrophil motility and attachment.

Polymorphonuclear neutrophils (PMN) traverse basement membrane to reach sites of infection. We have studied the role of laminin, a specific basement membrane component, in this process using three assay systems. In the Boyden chamber, laminin was found to stimulate chemotaxis of neutrophils while fibronectin did not. Co-incubation of cells with antibody to laminin blocked this chemotaxis, while antibody to fibronectin was without effect. In the human amnion system, neutrophils were shown to penetrate through the tissue when the peptide chemoattractant f-Met-Leu-Phe was placed on the opposing side. Antibody to laminin, but not to fibronectin, blocked this penetration. In an attachment assay system, laminin, but not fibronectin, was found to increase dispase-treated neutrophil attachment to type IV (basement membrane) collagen-coated plastic and to a plastic substrate itself. Electrophoretic analysis of PMN extract indicated the presence of laminin, and indirect immunofluorescence suggested that laminin is localized on the surface of the neutrophils. These data suggest that PMN can bind laminin on their cell surfaces, use laminin to attach to basement (type IV) membrane collagen, and migrate toward a gradient of laminin. These properties may be important for the passage of neutrophils from the circulation to sites of infection.

Amnion↗
PubMed1986

Chemotaxis of human gingival epithelial cells to laminin. A mechanism for epithelial cell apical migration.

Laminin, a large glycoprotein (Mr = 10(6)) and a major component of basement membrane, is shown here to be a potent chemoattractant for human gingival epithelial cells. Laminin stimulated chemotaxis and chemokinesis of gingival epithelial cells in the modified Boyden chamber assay. This effect appeared to be laminin receptor mediated. Gingival epithelial cells were shown to bind laminin (Kd = 2.0 nM) with 10,000 to 30,000 binding sites per cell. Antilaminin antibody, which inhibited laminin binding, inhibited the chemotactic response of epithelial cells to laminin, while antifibronectin was without effect. Fibronectin was not as potent a chemoattractant as laminin. Other biological response modifiers were also tested; of these, Type IV collagen and epidermal growth factor were active as chemoattractants, although not as effective in inducing chemotaxis as laminin. The data indicate that laminin and other components of basement membrane may be important in regulating the migration and growth of gingival epithelial cells.

Adult↗
PubMed1985

EGF induces receptor down-regulation with no receptor recycling in KB cells.

Several ligands, including epidermal growth factor (EGF), have been found to negatively modulate or down-regulate their specific plasma membrane receptors. Using both 125I-EGF and a monoclonal antibody against the EGF-receptor (EGF-R1), we studied the down-regulation of the EGF-receptor in the human adenocarcinoma cell line KB. The results presented here demonstrate that incubating KB cells at 37 degrees C with EGF rapidly decreases the number of plasma membrane EGF-receptors. In addition, there is a concomitant rise of equal magnitude in the number of EGF molecules taken up. The latter result argues strongly that there is negligible recycling of the EGF-receptor in KB cells and that the major portion of internalized EGF-receptor complexes are transported to lysosomes and subsequently degraded. The fate of the EGF-receptor is markedly different from that of receptors not subject to down-regulation. The biochemical signals that operate to regulate such diverse receptor traffic in cells remains to be elucidated.

Carcinoma↗
PubMed1985

Human endothelial cells are chemotactic to endothelial cell growth factor and heparin.

The response of human endothelial cell migration to various extracellular matrix components and growth factors has been assessed. Human endothelial cells demonstrate increased chemotaxis and chemokinesis when placed in a modified Boyden chamber with endothelial cell growth factor (ECGF) used at a concentration of 10(-9) M. Anti-ECGF antibody inhibits the chemotactic response. Heparin (10(-8) to 10(-10) M) was also chemotactic and was shown to potentiate the chemotactic activity of ECGF. Although laminin, fibronectin, the polypeptide (epidermal, fibroblast, and nerve) growth factors, and collagen types I, II, III, IV, and V demonstrate a chemotactic response, these activities were one third to one half less than observed with ECGF. These data suggest that ECGF and heparin may play a significant role as response modifiers of human endothelial cell migration which may be relevant to tumor metastasis, wound healing, and atherogenesis.

Cells, Cultured↗
PubMed1984

Down-regulation of the epidermal growth factor receptor in KB cells is due to receptor internalization and subsequent degradation in lysosomes.

Using a monoclonal antibody to the human epidermal growth factor (EGF) receptor (EGF-R1), we have followed the metabolism of the receptor and the pathway of its internalization in KB cells after the addition of EGF. Measurement of surface binding of 125I-labeled EGF showed that about 80% of EGF binding activity disappeared from the plasma membrane after a 10-min exposure to EGF at 37 degrees C. Immunoprecipitation of the receptor from [35S]methionine-labeled cell extracts with EGF-R1 showed that EGF caused the receptor to be degraded with a half-life of 40 min. Immunofluorescence using EGF-R1 showed an EGF-dependent redistribution of the EGF receptor. In cells not exposed to EGF, almost all of the receptor was diffusely distributed on the cell surface. After EGF addition, the receptor was rapidly internalized, first appearing in small punctate organelles characteristic of receptosomes and then in larger perinuclear lysosome-like structures. By 120 min almost all of the immunoreactive EGF receptor had disappeared from the cells. Immunocytochemistry at the electron microscopic level confirmed these light microscopic findings. The diffusely distributed receptor on the cell surface first clustered into clathrin-coated pits in the presence of EGF, next was internalized into receptosomes, appeared transiently in transreticular Golgi elements, and finally was seen in lysosomes. This EGF-dependent down-regulation and degradation of the EGF receptor in KB cells provides a striking example of ligand-dependent clustering and internalization of a receptor, followed by degradation in lysosomes of both ligand and receptor.

Antibodies, Monoclonal↗
PubMed1980

DNA-binding specificity of a chromatin non-histone protein fraction of HeLa cells.

The DNA-binding site of a previously characterized non-histone chromosomal protein antigen(s) from HeLa cells was investigated for its species specificity. Treatment with large amounts of micrococcal nuclease abolishes immunoactivity, which can then be recovered by the subsequent addition of human or HeLa DNA to reconstitute the immune complex. Neither rat nor calif DNA exhibits this property, but DNA from monkey cells gives considerable activity. The antigen is not, however, detectable in monkey chromatin.

Animals↗