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Biomedical subjects

A B Roberts

Publications and source records attributed to A B Roberts.

At least 289 records · Page 16Linked to original sources

Suppression of carcinogenesis by retinoids: interactions with peptide growth factors and their receptors as a key mechanism.

To understand the molecular mechanism of action of retinoids in control of differentiation and carcinogenesis, it is now necessary to consider the interactions of retinoids with oncogenes, as well as with peptide growth factors and their receptors. Experimental studies in these areas are described. In particular, it is shown that retinoic acid inhibits the proliferative effects of transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) on fibroblasts that have been transfected with the c-myc oncogene. The ability of retinoic acid to induce differentiation in several types of human and murine tumor cells also is associated with the ability of retinoic acid to suppress the expression of either the c-myc oncogene, or the related N-myc gene.

Animals↗

Transforming growth factor-beta controls receptor levels for epidermal growth factor in NRK fibroblasts.

NRK fibroblasts exposed to transforming growth factor-beta (TGF-beta) show increased binding of radiolabeled epidermal growth factor (EGF) relative to untreated cells. The binding of another growth factor, rat insulin-like growth factor-II, is unaffected. The increase in EGF binding induced by TGF-beta is not due to inhibition of EGF processing nor to an alteration in the affinity of plasma membrane EGF receptors. However, treatment of the cells with TGF-beta does cause a rapid increase in the number of plasma membrane receptors for EGF. TGF-beta has little effect on the rate of overall protein synthesis, but the increase it induces in EGF binding can be completely inhibited by cycloheximide and tunicamycin. Thus a selective synthetic mechanism underlies TGF-beta action. Cells incubated with TGF-beta also show altered down regulation of their EGF receptors in response to the ligand; concentrations of EGF that can induce strong biological responses no longer decrease the plasma membrane receptor level below the basal state. These results agree well with the known specificity and synergism of the interaction between TGF-beta and EGF. Moreover, they describe a mechanism of growth control in which bioactive peptides act coordinately through a regulatory effect on the number of cell-surface receptors.

Animals↗

Human transforming growth factor-alpha: precursor structure and expression in E. coli.

Transforming growth factor-alpha (TGF-alpha) is secreted by many human tumors and can induce the reversible transformation of nontransformed cell lines. Using long synthetic deoxyoligonucleotides as hybridization probes we isolated an exon coding for a portion of TGF-alpha from a human genomic DNA library. Utilizing this exon as a probe, a cell line derived from a human renal cell carcinoma was identified as a source of TGF-alpha mRNA. A cloned TGF-alpha cDNA was isolated from a cDNA library prepared using RNA from this cell line, and was found to encode a precursor polypeptide of 160 amino acids. The 50 amino acid mature TGF-alpha produced by expression of the appropriate coding sequence in E. coli binds to the epidermal growth factor receptor and induces the anchorage independence of normal mammalian cells in culture.

Adenocarcinoma↗

Antagonistic actions of retinoic acid and dexamethasone on anchorage-independent growth and epidermal growth factor binding of normal rat kidney cells.

Type beta transforming growth factor (TGF-beta), in combination with epidermal growth factor (EGF) can induce nonneoplastic normal rat kidney cells to express a transformed phenotype and to form colonies in soft agar. Retinoic acid by itself has no effect on colony formation; but at concentrations of 10(-9) M or greater, it can greatly enhance the response of the cells to EGF and TGF-beta, as measured by colony growth in soft agar and expression of a transformed morphology in monolayer culture. Dexamethasone, at concentrations above 10(-9) M, has an opposite effect, inhibiting the TGF-beta-dependent formation of colonies in soft agar and restoring a more normal morphology to the cells. Added simultaneously, these two modulators act antagonistically; at equimolar concentrations, their opposite effects on colony formation are canceled. Retinoic acid and dexamethasone also have opposite and antagonistic effects on the binding of 125I-labeled EGF to normal rat kidney cells. Retinoic acid enhances the binding of EGF up to 6-fold, while dexamethasone reduces the binding to 50 to 60% of control levels. These effects on EGF binding show a dose dependence similar to the effects on colony formation in soft agar and on morphology in monolayer culture. Optimal effects on binding are observed 40 to 60 hr after treatment of the cells. It can be concluded that the abilities of retinoic acid and dexamethasone to alter expression of the transformed phenotype induced by treatment of normal rat kidney cells with TGF-beta and EGF are mediated at least in part through their effects on the EGF receptor.

Animals↗

Purification and properties of a type beta transforming growth factor from bovine kidney.

Type beta transforming growth factor (TGF-beta) has been purified 200 000-fold from bovine kidneys. This peptide is characterized by its ability to induce anchorage-dependent normal rat kidney cells to grow in soft agar in the presence of epidermal growth factor (EGF); TGF-beta is not mitogenic for cells grown in monolayer culture. Purified TGF-beta does not compete with EGF for binding to membrane receptors. The concentration of TGF-beta required to elicit a half-maximal response for formation of colonies greater than 3100 micron2 in the soft agar assay is 2-3 pM (55 pg/mL) when assayed in the presence of 0.8 nM EGF (5 ng/mL). The four-step purification procedure which includes chromatography of acid--ethanol tissue extracts on polyacrylamide sizing gels, cation exchange, and two steps of high-pressure liquid chromatography results in a 10% overall yield of colony-forming activity with a recovery of 3-4 micrograms/kg. Amino acid analysis of purified TGF-beta shows 16 half-cystine residues per mole. Analysis of the purified polypeptide by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicates that TGF-beta is composed of two closely related polypeptide chains cross-linked by disulfide bonds. In the absence of beta-mercaptoethanol, the colony-forming activity is associated with a single silver-staining band of molecular weight 25 000; in the presence of beta-mercaptoethanol, the TGF-beta is converted to an inactive species that migrates as a single band of molecular weight 12 500-13 000. Sequence analysis indicates that at least the first 15 N-terminal amino acids of the two TGF-beta subunits are identical.

Amino Acid Sequence↗

Fructosamine in diabetic pregnancy.

Fructosamine, an indicator of glycosylated serum protein, was measured in 79 non-diabetic pregnant women and 20 women with gestational diabetes. The test provided a clear discrimination between groups; it detected 17 (85%) of the women with gestational diabetes and gave only 4 (5%) false-positive results. 19 women with established diabetes before pregnancy had very high levels. Maternal fructosamine at 29 weeks' gestation correlated significantly with both fasting blood glucose levels at the time and birthweight ratio. Levels of fructosamine in cord blood were significantly higher in gestational diabetic than in normal pregnancies, suggesting a possible additional role for fructosamine in retrospectively detecting the hyperglycaemic fetus. Fructosamine estimation is fully automated and may provide a simple, inexpensive means to screen for diabetes in pregnancy.

Adult↗

Polypeptide transforming growth factors isolated from bovine sources and used for wound healing in vivo.

Transforming growth factors, which are polypeptides that induce the transformed phenotype in nonneoplastic cells, have been isolated in bulk amounts from bovine salivary gland and kidney. In experiments in which wound healing chambers were implanted subcutaneously in the backs of rats, these bovine transforming growth factors accelerated the accumulation of total protein, collagen, and DNA in treated chambers. These studies thus show an effect of an isolated transforming growth factor in vivo.

Animals↗

Sarcoma growth factor from conditioned medium of virally transformed cells is composed of both type alpha and type beta transforming growth factors.

Sarcoma growth factor (SGF) derived from conditioned medium of Moloney sarcoma virus-transformed cells and partially purified by gel filtration (crude SGF) has been characterized by its ability both to compete with epidermal growth factor (EGF) for binding to membrane receptors and to induce anchorage-independent growth of untransformed cells. We now show that further purification of crude SGF by reverse-phase HPLC on muBondapak C18 and CN columns at pH 2 resolves it into two distinctly different polypeptides, which we call types alpha and beta transforming growth factors (TGFs). Type alpha TGF (TGF-alpha), but not type beta TGF (TGF-beta), competes for binding to the EGF receptor and induces the formation of small colonies (1,000-2,000 micron2) of normal rat kidney cells in soft agar. Both TGF-beta and EGF or TGF-alpha must be present in order to induce the formation of large colonies (7,000-15,000 micron2). Based on EGF competing equivalents as determined from a radioreceptor assay with 125I-labeled EGF in normal rat kidney cells, the relative ability of EGF and TGF-alpha to potentiate TGF-beta-dependent colony formation is in the order conditioned-medium TGF-alpha greater than EGF greater than intracellular TGF-alpha. Suboptimal concentrations of the same polypeptides give additive potentiation of the TGF-beta-dependent colony-forming response; saturating levels potentiate a similar maximum response whether used alone or in various combinations. The data indicate that the EGF-competing activity of crude SGF is due to its TGF-alpha component alone, whereas the soft-agar colony-forming activity is due to the combined action of two distinct polypeptides, TGF-alpha and TGF-beta.

Animals↗

Transforming growth factors from neoplastic and nonneoplastic tissues.

Transforming growth factors (TGFs) are a heterogeneous family of polypeptides that induce anchorage-independent growth in nonneoplastic anchorage-dependent cells. They have been found in many tissues, both neoplastic and nonneoplastic. All TGFs isolated thus far are of low molecular weight (6000-25,000), are acid and heat stable, and are inactivated by reagents that reduce disulfide bonds. TGFs have been classified as type alpha or type beta based on their interactions with the receptor for epidermal growth factor (EGF) and their requirement for EGF (or an EGF-like polypeptide) for functional activity. TGF-alpha and TGF-beta act synergistically. TGF-alpha induces phosphorylation of tyrosine in the EGF receptor. TGF-beta, isolated from bovine sources, accelerates experimental wound healing in rats.

Animals↗

Selective accumulation of low density lipoproteins in damaged arterial wall.

To determine whether damaged arterial wall selectively accumulates lipoproteins, normocholesterolemic rabbits were injected with human radiolabeled low density lipoproteins, high density lipoproteins, and/or albumin 24 hr to 12 weeks after balloon-catheter de-endothelialization of the abdominal aorta. When 125I-labeled low density lipoproteins and 99mTc-labeled albumin were injected simultaneously, the amount of 125I-low density lipoprotein present 24 hr later in abdominal aortas increased steadily, for several weeks, above the amount present at 24 hr in control animals. The increase correlated closely with the degree of re-endothelialization and correlated closely with the degree of re-endothelialization and reached an average maximum for the whole abdominal aorta of three times control when re-endothelialization was between 75 and 85% complete. By contrast, the amounts of 99mTc-albumin or 125I-labeled high density lipoprotein in balloon-damaged abdominal aortas, and the amounts of 125I-low density lipoprotein, 125I-high density lipoprotein, or 99mTc-albumin in undamaged thoracic aortas of injured animals showed no such increase. As early as 2 weeks after de-endothelialization, en face radioautographs made following injection of 125I-labeled low density lipoproteins revealed localized areas of greatest radioactivity around the leading edges of regenerating endothelial islands, broad areas of intermediate radioactivity corresponding to the de-endothelialized areas, and very like radioactivity in the re-endothelialized areas. This pattern occurred rarely with 125I-labeled high density lipoproteins and not at all with 125I-labeled albumin. The results suggest that low density lipoproteins are selectively accumulated by the healing rabbit aorta and that the accumulation is greatest in regions where the endothelium is actively regenerating.

Albumins↗

Imaging of acute arterial injury with 111In-labeled platelets: a comparison with scanning electron micrographs.

The relationship between degree of acute arterial injury, extent of platelet deposition, and ability to visualize arterial injury with Indium 111-labeled platelets was studied in 18 rabbits. An aortic lesion was made with a balloon catheter in each animal immediately after injection of autologously labeled platelets. Three nonlesioned rabbits with 111In-labeled platelets served as controls. An additional control study was performed in 12 lesioned rabbits in which nine were injected with 111In-labeled plasma protein and three with 111In-labeled red blood cells. A postmortem scanning electron microscope study of the aortae was made to determine the degree of injury to the intima and the amount of platelet deposition on the damaged arterial wall. The radionuclide scans and scanning electron micrographs were then compared. Lesions were seen in ten of 18 animals with labeled platelets that had extensive regions of denuded endothelium covered by a contiguous layer of platelets. Lesions consisting of patchy deendothelialization and platelet deposition could not be visualized on the scans. Red blood cells and fibrin were not conspicuous on micrographs of the lesions. No lesions were visualized in animals receiving 111In-labeled plasma protein or red blood cells before arterial injury, despite platelet deposition in the lesions.

Animals↗

Synergistic interaction of two classes of transforming growth factors from murine sarcoma cells.

Transforming growth factors (TGFs) isolated from murine sarcoma virus-transformed 3T3 cells have been separated by high-pressure liquid chromatography into two subsets. One subset, called TGF alpha, competes with epidermal growth factor (EGF) for receptor sites, whereas the other, called TGF beta, does not. TGB beta, purified by high-pressure liquid chromatography, will not induce formation of large colonies of cells in soft agar in the absence of TGF alpha or EGF. However, the combined action of either TGF alpha or EGF (which by themselves are relatively ineffective in promoting growth of cells in soft agar) together with TGF beta results in a potent synergistic effect, with formation of large colonies. Chemically modified analogs of EGF also potentiate TGF beta activity to the extent that they bind to the EGF receptor. It is suggested that TGF beta may be an important mediator of the known effects of both TGF alpha and EGF on neoplastic transformation.

Animals↗