Epidermal growth factor receptor cytoplasmic domain mutations trigger ligand-independent transformation.

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.

Ligand activation of overexpressed epidermal growth factor receptors transforms NIH 3T3 mouse fibroblasts.

The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human EGF receptor without structural alterations. Activation of overexpressed normal receptor alone appears to be sufficient for transformation of NIH 3T3 cells in vitro.

Characterization of murine monoclonal antibodies directed against basic fibroblast growth factor.

Monoclonal antibodies (McAbs) were developed that identify the complete (1-146 aa) and the NH2-terminal truncated (des 1-15) form of bovine basic fibroblast growth factor (bFGF). Four McAbs, designated McAbs 6, 8, 38, and 42, bind the complete form of bFGF found in bovine pituitary, brain, and adrenal gland. One of these McAbs, McAbs 42, also binds to the des 1-15 aa form of bFGF found in bovine adrenal gland, kidney, and corpus luteum. None of the McAbs binds bovine-brain-derived acidic FGF (aFGF). McAbs 6, 8, and 38 recognized the same epitope located within the first ten residues of the NH2-terminal of complete bFGF. McAb 42 recognizes a "core" epitope found on both the complete and des 1-15 aa bFGFs. The McAbs are murine IgGs with affinity constants of 10(7)-10(8) liter/M for bovine-pituitary-derived bFGF. McAbs 8 and 42 have been used in a two-site ELISA to detect the complete form of bFGF. The ELISA is sensitive to 38.5 fmole/well of bFGF and is not affected by the presence of calf serum or bovine-brain-derived aFGF. These McAbs should be useful in distinguishing the native and des 1-15 aa forms of bFGF from each other, and from aFGF and other growth factors.

Effect of retina-derived basic and acidic fibroblast growth factor and lipoproteins on the proliferation of retina-derived capillary endothelial cells.

Retina-derived capillary endothelial (RCE) cells have been established in culture, taking advantage of their ability to proliferate at clonal density when maintained on extracellular matrix (ECM)-coated dishes in the presence of serum-supplemented medium. This cell type formed at confluence a monolayer of small, tightly packed, contact inhibited cells which expressed Factor VIII-related antigen. Both retina-derived basic and acidic fibroblast growth factors (FGF) were mitogenic when RCE cells were maintained on gelatin-coated dishes and exposed to serum-supplemented medium. Half-maximal stimulation of cell proliferation was observed with concentrations of 13 pg ml-1 for basic FGF and 2.5 ng ml-1 for acidic FGF. Maintaining RCE cells on extracellular matrix (ECM)-coated dishes greatly reduced their requirement for EGF in order to proliferate actively. Heparin strongly reduced the proliferative response of RCE cells to either basic or acidic FGF, as well as their response to serum alone, regardless of whether cells were maintained on gelatin or on ECM-coated dishes. When RCE cells maintained on ECM-coated dishes were exposed to defined medium, high density lipoproteins, transferrin, and FGF were required in order for these cells to proliferate actively.

Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex, and corpus luteum capillaries.

Bovine adrenal and brain cortex and corpus luteum-derived capillary endothelial cells have been established in culture, taking advantage of their ability to proliferate at clonal density when maintained on extracellular matrix (ECM) coated dishes in the presence of serum supplemented medium. All three cell types formed at confluency a monolayer of small, tightly packed, contact inhibited cells that express factor VIII related antigen. Their proliferative response to basic and acidic FGF when cells were maintained on plastic and exposed to serum supplemented medium was similar to that previously reported for endothelial cells derived from large vessels, with acidic FGF being 30-fold less potent than basic FGF. Their requirement for high density lipoproteins and transferrin in order to proliferate actively when maintained on ECM-coated dishes and exposed to serum-free conditions was also similar to that previously reported for endothelial cells derived from large vessels. Heparin strongly reduced the proliferative response of capillary endothelial cells to either basic or acidic FGF, as well as their response to serum alone, regardless of whether cells were maintained on plastic or on ECM-coated dishes. The present data indicate that bovine endothelial cells derived from large or small vessels are indistinguishable in so far as their response to growth factors, plasma factors, and substrata are concerned.

Effect of lipoproteins and growth factors on the proliferation of BHK-21 cells in serum free culture.

Baby hamster kidney-derived cells (BHK-21 cell line), seeded at low density on gelatin coated dishes and exposed to a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium, proliferate actively when exposed to high density lipoproteins (HDL), transferrin, and basic or acidic fibroblast growth factor (FGF). This serum free medium combination supported cell multiplication at a rate equal to that of serum supplemented medium, and at low cell input (10(3) cells/35-mm dish). Epidermal growth factor (EGF), although mitogenic for BHK-21 cells, was less efficient than either basic or acidic FGF in supporting cell growth. When the potency of basic and acidic FGF were compared, acidic FGF was 10-fold less potent than basic FGF. The requirement of BHK-21 cells for transferrin appears to be minimal since cells exposed to HDL and basic FGF could be serially transferred for at least 50 cumulative population doublings in the absence of transferrin.

Isolation of pituitary fibroblast growth factor by fast protein liquid chromatography (FPLC): partial chemical and biological characterization.

Bovine pituitary fibroblast growth factor has been purified 222,000-fold to homogeneity by a combination of differential salt extraction, gel filtration, and ion exchange chromatography on Mono S column. Pituitary FGF is a single-chain polypeptide with an apparent molecular mass of 15,800 and an isoelectric point of 9.6. It is highly active in triggering the proliferation of bovine and human vascular endothelial cell [half-maximal stimulation at 23-40 pg/ml (1.5-2.6 pM) and saturation between 140 and 280 pg/ml (9.3-18.6 pM)]. It displays a similar activity on bovine vascular smooth muscle cells, corneal endothelial cells, granulosa and adrenal cortex cells, and rabbit costal chondrocytes.

Comparison of the ability of basement membranes produced by corneal endothelial and mouse-derived Endodermal PF-HR-9 cells to support the proliferation and differentiation of bovine kidney tubule epithelial cells in vitro.

The proliferation and morphological differentiation of bovine kidney collecting-tubule epithelial cells has been examined as a function of substrata and plasma factors. Collecting kidney tubule explant maintained in vitro gave rise to two distinct cell populations; one was composed mostly of fibroblastic cells whereas the other was epithelioid (EP cells). The proliferation of fibroblastic cells when exposed to serum-supplemented medium was best expressed when cells were maintained on a basement membrane produced by bovine corneal endothelial cells. This basement membrane has a composition, which in previous studies has been shown to favor the proliferation of mesenchymal cells. In contrast, the proliferation of EP cells was best expressed when cells were maintained on a basement membrane produced by the mouse-derived endodermal cell line PF-HR-9 (HR-9-BM). This basement membrane has a biochemical composition very similar to the basement membrane underlying the kidney tubules. Although the fibroblast confluent monolayer maintained on bovine corneal endothelial cell extracellular matrix did not undergo morphogenesis, the confluent monolayer of EP cells maintained on HR-9-BM shows hemicyst formation, suggesting that they were capable of vectorial fluid transport. They also built a complex three-dimensional kidney tubulelike network. Some tubules became grossly visible and floated into the tissue culture medium, remaining tethered to the cell monolayer at either end of the tubule. On an ultrastructural level, the tubules consisted of cells held together with junctional complexes arranged so as to form a lumen. The smallest lumen were bordered by 2-3 cells, and the largest ones by 8-15 cells. The lumens of the larger tubules did contain granular fibrillar and amorphous debris. Low-density EP cell cultures maintained on HR-9-BM could be induced to proliferate at a rate approaching that of cultures exposed to serum when they were exposed to medium supplemented with high-density lipoprotein (HDL, 750 micrograms protein/ml) and transferrin (50 micrograms/ml). When exposed to HDL concentrations equal or lower than 250 micrograms protein/ml, low-density cultures proliferated at a slow rate and readily formed tubulelike structures. This observation indicates that EP cells do not need to reach confluence to undergo morphogenesis, and that HDL, which in the presence of transferrin supports the cell proliferation, can favor their differentiation into tubulelike structures once its concentration becomes limiting for mitogenesis.

Stimulation of the proliferation of the Madin-Darby canine kidney (MDCK) epithelial cell line by high-density lipoproteins and their induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

MDCK Cells seeded on extracellular matrix- (ECM) coated dishes and exposed to medium supplemented with high-density lipoproteins (HDLs, 750 micrograms protein/ml) and transferrin (10 micrograms/ml) have a proliferative rate, final cell density, and morphological appearance similar to those of cells grown in serum-supplemented medium. The mitogenic stimulus provided by HDLs is not limited by the initial cell density at which cultures are seeded, nor is it limited in time, since cells grown in medium supplemented with transferrin and HDLs grew to at least 50 generations. The presence of HDLs in the medium is required in order for cells to survive, since cells actively proliferating in the presence of medium supplemented with HDLs and transferrin begin to die within 2 days after being transferred to medium supplemented only with transferrin. Low-density lipoprotein (LDL) is mitogenic for MDCK cells when present at low concentrations (from 2.5 to 100 micrograms protein/ml). Above 100 micrograms protein/ml, LDL is cytotoxic and therefore cannot support cell proliferation at an optimal rate. The mitogenic effect of HDLs is also observed when cells are maintained on fibronectin-coated dishes. However, the proliferative rate of the cells is suboptimal and cultures cannot be passaged on this substrate indefinitely, as they can be on ECM-coated dishes. A close association between the ability of HDLs to support cell proliferation and their ability to induce the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is observed. HMG CoA reductase activity is 18 times higher (70 pmoles/min/10(6) cells) in proliferating cells than in confluent, nondividing cells (4 pmoles/min/10(6) cells). The HMG Coa reductase activity of sparse cells is more sensitive to induction by HDLs (eight-fold higher than control cells) than is the enzyme activity of confluent cells (two-fold higher than control levels). The dose-response relationship between the abilities of HDLs to support proliferation and to induce HMG CoA reductase activity are similar. The time course of the stimulation of proliferation and the increase in enzyme activity of sparse, quiescent cells after exposure to HDLs are parallel. The HMG CoA reductase activity of sparse MDCK cells is induced six-fold by exposure to compactin, a competitive inhibitor of HMG CoA reductase. This induction of HMG CoA reductase is prevented by mevalonic acid, not affected by LDL, and synergistically enhanced by simultaneous exposure to HDLs. HDLs effect a rescue from the cytotoxic effect of compactin, whereas LDL does not.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurite outgrowth and protein synthesis by PC12 cells as a function of substratum and nerve growth factor.

Numerous studies have implied that enhanced cell-substratum adhesion plays a role in neurite outgrowth by neuronal cells. Using an extracellular matrix (ECM) produced by cultured corneal endothelial cells, we have investigated attachment and de novo neurite outgrowth by the pheochromocytoma cell line, PC12. PC12 cells were found to attach more rapidly and efficiently to the ECM than to plastic or collagen-coated surfaces. An extensive but temporary (5- to 10-day) neurite outgrowth occurred in the absence of nerve growth factor (NGF) when cells were on the ECM. However, long term neurite survival and further elongation required NGF. Our findings are consistent with the hypothesis that protein(s) in the ECM has an important role in neurite outgrowth. Thus, NGF may not so much initiate neurite outgrowth as it stabilizes neurites. ECM and NGF also were found to modulate cellular protein synthesis by PC12 cells. On ECM, a decreased synthesis of many high molecular weight proteins (Mr greater than 85,000) was observed in comparison to that of cells on collagen-coated dishes. The presence of neurites (in the presence of absence of NGF) as associated with the induction of the synthesis of a cellular protein (Mr = 55,000 to 56,000 and pI of 5.6). NGF was found to increase markedly the synthesis of two secreted proteins, while it drastically reduced the synthesis of all others regardless of the substratum upon which the cells were maintained.

High density lipoproteins and the growth of vascular endothelial cells in serum-free medium.

Low density bovine vascular endothelial cell cultures maintained on dishes coated with an extracellular matrix can be grown in serum-free Dulbecco's modified Eagle's medium supplemented with high density lipoprotein (HDL) and transferrin. Such cultures do not require insulin. Early passage cultures exposed to HDL and transferrin grew as well as cultures exposed to optimal serum concentrations and could be passaged repeatedly in total absence of serum. A requirement for fibroblast growth factor to ensure an optimal growth could be observed only with late-passage cultures. The present results suggest strongly that HDL is involved in supporting the proliferation of vascular endothelial cells in vitro. This may be important for our understanding of the biological role of HDL "in vivo."