Radioimmunoassay of factor V in human plasma and platelets

Homogeneous, single-chain human factor V was used to develop a double antibody competition radioimmunoassay to measure factor V concentrations in plasma and platelets. Standard curves were constructed that allow for the detection of as little as 20 ng factor V/ml of plasma. Normal factor V concentrations range from 4 to 14 micrograms/ml of plasma with an average value of 7.0 +/- 2.0 micrograms/ml (n = 64). No correlation was observed between antigen levels and age or sex. The radioimmunoassay data are consistent with factor V clotting assays, providing freshly drawn plasma is used in the bioassay. Radioimmunoassay of washed platelets indicate that 0.63-1.93 microgram of factor V is present per 2.5 X 10(8) platelets (4612-14128 molecules of the factor V platelet). When normalized to individual hematocrits and platelet count, the data indicated that platelets contribute approximately 18%-25% of the factor V found in whole blood. In addition, two individuals with functionally deficient factor V were examined and found to be deficient in both antigen and activity.     

Adipose conversion of 3T3 cells depends on a serum factor.

The adipose conversion of 3T3-F442A cells depends on an adipogenic factor in serum. In the presence of this factor, cells grown to confluence become spherical, greatly increase the activity of their lipogenic enzymes, and accumulate triglyceride. In the absence of the adipogenic factor, the cells grow normally, but when they reach confluence they do not become spherical, do not accumulate triglyceride, and do not undergo any increase in activity of lipogenic enzymes. In cattle there is a great deal more of the adipogenic factor in the serum before birth than in the serum of grown animals. The nature of the adipogenic factor suggests that it may play an important role in the development of adipose tissue.     

Mouse hepatic endothelial cells in culture secrete a growth inhibitor for hepatic lipocytes and Balb/c 3T3 fibroblasts

Hepatic lipocytes are sinusoidal cells in close contact to endothelial cells. They proliferate, switch to a fibroblastic phenotype and synthetize collagen during hepatic fibrosis. Since it is known that vascular endothelial cells can influence the proliferation of neighboring cells such as smooth muscle cells, we investigated the role of hepatic endothelial cells on the growth of lipocytes and Balb/c 3T3 fibroblasts. Concentrated conditioned medium from endothelial cells inhibited both [3H]thymidine incorporation and actual growth of lipocytes and Balb/c 3T3 fibroblasts. The inhibition was lost when conditioned medium was treated with heat or trypsin, or when medium was conditioned in the presence of cycloheximide. We conclude that hepatic endothelial cells secrete a proteic growth inhibitor for lipocytes and hepatic Balb/c 3T3 fibroblasts. This inhibitor could be of importance in limiting lipocyte proliferation in the liver and possibly in preventing hepatic fibrosis.     

Isolation of a cationic polypeptide from human serum that stimulates proliferation of 3T3 cells.

A basic polypeptide that stimulates DNA synthesis and cell division in confluent populations of mouse Balb/c-3T3 cells has been isolated from whole human serum, and has been separated from the heterogenous group of molecules with insulin-like activity. This highly purified basic polypeptide has a molecular weight of 1.3 x 10(4) and an isoelectric point of 9.7. Approximately 10(7) polypeptide molecules in the growth medium allow the replication of one density-inhibited cell.     

Specific binding to cultured cells of 125I-labeled type beta transforming growth factor from human platelets.

Purified type beta transforming growth factor from human platelets (TGF beta) radioiodinated with 125I-labeled Bolton and Hunter reagent was found to bind to a variety of cultured cells of both epithelial and mesenchymal origin, including normal human fibroblasts and keratinocytes. TGF beta binding sites have also been found on three mouse embryo-derived fibroblast-like cell lines with lower levels of TGF beta binding on the chemically transformed derivatives of these cell lines. A variety of human tumor cell lines was shown to have an inverse correlation between their level of TGF beta binding and their ability to form colonies in soft agar. The mouse embryo-derived AKR-2B (clone 84A) cells reached maximal binding of 125I-labeled TGF beta after 2 hr at 22 degrees C. Scatchard analysis of the equilibrium binding of TGF beta to AKR-2B (clone 84A) cells gives a Kd of 33 pM with approximately equal to 10,500 binding sites per cell. This Kd for TGF beta binding to AKR-2B (clone 84A) cells agreed well with the ED50 of 40 pM for stimulation of colony formation of these cells by TGF beta. The TGF beta binding sites on the AKR-2B cells were shown to be specific for TGF beta with no significant competition with epidermal growth factor, fibroblast growth factor, or insulin and only a small level of competition with high concentrations of platelet-derived growth factor. Partially purified preparations with TGF beta-like activity from mouse embryos and medium conditioned by mouse embryo-derived cells competed effectively for binding to the TGF beta receptor.     

Enhanced synthesis and stabilization of Mr 68,000 protein in transformed BALB/c-3T3 cells: candidate for restriction point control of cell growth.

We have proposed that transformation of cells to tumorigenicity by chemical carcinogens can depend upon stabilization of a protein responsible for growth regulation. Cell kinetic experiments in which normal and benzo[a]pyrene-transformed BALB/c-3T3 cells were pulsed with cycloheximide indicated this protein should have a half-life of a few hours in normal cells and be considerably more stable in transformed cells [Campisi, J., Medrano, E. E., Morreo, G. & Pardee, A. B. (1982) Proc. Natl. Acad. Sci. USA 79, 436-440]. A protein with these properties has not yet been reported. We have searched for such a protein using two-dimensional electrophoresis to resolve protein from cells labeled with [35S]methionine. Among approximately 1,000 proteins that were resolved in these gels, we have found one that has a greater rate of synthesis and stability in benzo[a]pyrene-transformed than in untransformed cells. This result satisfies a necessary prediction of our labile protein hypothesis. We suggest that this protein could be important in determining the loss of growth regulation in these tumor cells.     

Expression of human epidermal growth factor precursor cDNA in transfected mouse NIH 3T3 cells.

Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 microM ZnCl2. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approximately 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with 125I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.     

Characterization of material with epidermal growth factor immunoreactivity in human serum and platelets

We investigated the molecular nature of the epidermal growth factor immunoreactivity (ir-EGF) in serum and platelets of normal subjects. In serum, ir-EGF appeared and increased during spontaneous blood coagulation, reaching a plateau in 2 h. The mean plateau measured by time-resolved immunofluorometric assay was 778 pg/mL [130 pmol/L; range, 465-1352 pg/mL (78-225 pmol/L)] for men (n = 66), and 774 pg/mL (129 pmoL/L; range, 521-1114 pg/mL (87-186 pmol/L)] for women (n = 33). Serum samples (n = 9), when examined under nonreducing conditions by high performance liquid chromatography, contained three mol wt components. Their approximate proportions averaged 56 +/- 6% (+/- SD) for a 140K component, 22 +/- 9% for a 67K component, and 22 +/- 6% for a component coeluting with the 6K EGF standard. The molecular size distribution of ir-EGF released from isolated platelets varied with the treatment of the platelets. After stimulation with a Ca2+ ionophore, the proportions of the 140K, 67K, and 6K components were 61 +/- 11%, 20 +/- 8%, and 17 +/- 4% (n = 5), respectively, as in serum. In addition, we found a small amount, (approximately 1%) of a 17K component. When platelets (n = 6) were ruptured by sonication and repeated freeze-thawing, the 67K component formed 53 +/- 7% of the total; the proportions of the 140K, 17K, and 6K components were 14 +/- 7%, 2.1 +/- 0.7%, and 31 +/- 4%, respectively. Under reducing conditions the 17K and 6K components remained intact, but part of the 140K component and all of the 67K component were cleaved to a 35-37K form. After protease digestion of the higher mol wt components, the ir-EGF was exclusively of the 6K form. The 67K and 6K components bound to the EGF receptor, whereas the 140K component bound inconsistently. We conclude that treatment of platelets with Ca2+ ionophore produces ir-EGF components similar to those found in incubated serum but different from those obtained by freeze-thawing. The 67K mol wt component appears to be the main storage form of EGF in platelets; at least two independent mechanisms appear to exist for EGF release.     

Colchicine inhibits epidermal growth factor degradation in 3T3 cells.

Colchicine (2 microM) did not affect the initial rate of association of 125I-labeled epidermal growth factor (125I-EGF) to Swiss 3T3 cells but continued incubation (up to 24 hr) led to an increase in cell-associated radioactivity. The effect is also produced by Colcemid, vinblastine, and podophyllotoxin but not by lumicolchicine. Disruption of microtubules with colchicine does not alter the rate of "down regulation" of EGF receptors, suggesting the binding and internalization of the factor proceed unchanged. However, colchicine markedly decreases the rate of appearance of acid-soluble radioactivity from cells either incubated continuously with 125I-EGF for 24 hr or exposed to the radioactive peptide for only 1 or 3 hr. The results indicate that colchicine decreases the rate of degradation of internalized 125I-EGF. Because antitubulin agents enhance the mitogenic effect of EGF our results suggest that peptide degradation can be dissociated from the long-term biological effect.     

Invasiveness and metastasis of NIH 3T3 cells induced by Met-hepatocyte growth factor/scatter factor autocrine stimulation.

The met protooncogene product, Met, is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). NIH 3T3 cells express HGF/SF endogenously and become tumorigenic in nude mice via an autocrine mechanism when murine Met is expressed ectopically (Metmu cells) or when human Met and human HGF/SF are coexpressed (HMH cells). Here, we show that Metmu and HMH cells are invasive in vitro and display enhanced protease activity necessary for the invasive phenotype. In experimental and spontaneous metastasis assays, Metmu or HMH cells metastasize to the lung, but lower numbers of subcutaneously injected Metmu and HMH cells produced invasive tumors in the heart, diaphragm, salivary gland, and retroperitoneum. It has been reported elsewhere that Met expression increased with tumor passage in athymic nude mice, and these tumor explants show enhanced activity in the metastasis assays. Autocrine-mediated Met-HGF/SF signal transduction in NIH 3T3 mesenchymal cells may provide an important system for understanding the biological process of metastasis.     

Heat shock induces the release of fibroblast growth factor 1 from NIH 3T3 cells.

Fibroblast growth factor 1 (FGF-1) is a potent angiogenic and neurotrophic factor whose structure lacks a classical signal sequence for secretion. Although the initiation of these biological activities involves the interaction between FGF-1 and cell surface receptors, the mechanism responsible for the regulation of FGF-1 secretion is unknown. We report that murine NIH 3T3 cells transfected with a synthetic gene encoding FGF-1 secrete FGF-1 into their conditioned medium in response to heat shock. The form of FGF-1 released by NIH 3T3 cells in response to increased temperature (42 degrees C, 2 hr) in vitro is not biologically active and does not associate with either heparin or the extracellular NIH 3T3 monolayer matrix. However, it was possible to derive biologically active FGF-1 from the conditioned medium of heat-shocked NIH 3T3 cell transfectants by ammonium sulfate fractionation. The form of FGF-1 exposed by ammonium sulfate fractionation is similar in size to cytosolic FGF-1 and can bind and be eluted from immobilized heparin similarly to the recombinant human FGF-1 polypeptide. Further, the release of FGF-1 by NIH 3T3 cell transfectants in response to heat shock is reduced significantly by both actinomycin D and cycloheximide. These data indicate that increased temperature may upregulate the expression of a factor responsible for the secretion of FGF-1 as a biologically inactive complex that requires an activation step to exhibit the biological activity of the extracellular polypeptide mitogen.     

Release of platelet-derived growth factor from human platelets by arachidonic acid

Platelet alpha-granules contain a factor that stimulates the proliferation of arterial smooth muscle cells and may play a role in atherogenesis. We have studied the role of arachidonic acid in mediating the release of the platelet-derived growth factor (PDGF) from human platelets. PDGF was assayed by stimulating of [(3)H]thymidine incorporation into DNA of mouse 3T3 cells. Platelet aggregation and the release of platelet factor 4,beta-thromboglobulin, and serotonin were also studied. A biphasic response pattern was observed when gel-filtered platelets were incubated with arachidonate over the concentration range 0.01-0.4 mM. At low arachidonate levels (approximately 0.025-0.1 mM), specific concentration-dependent aggregation and release of PDGF and of the other components were observed. This effect was not seen with any of five other fatty acids tested and was suppressed by indomethacin (25 muM). At higher arachidonate concentrations (approximately 0.15-0.35 mM), a concentration-dependent turn-off of both aggregation and release occurred. At these concentrations the platelets remained functional, and no release of lactate dehydrogenase was observed. A similar biphasic pattern of arachidonate-induced aggregation and release was observed with platelet-rich plasma, over a similar range of arachidonate to albumin mole ratios. These studies demonstrate that PDGF and other alpha-granule constituents can be released from platelets specifically by arachidonate via an indomethacin-sensitive pathway, most probably involving the platelet cyclooxygenase and conversion of arachidonate to prostaglandin metabolities. The mechanisms responsible for the turn-off of the specific arachidonate-mediated responses at higher arachidonate concentrations remain to be defined.     

Generation of hematopoietic colony-forming cells from embryonic stem cells: synergy between a soluble factor from NIH-3T3 cells and hematopoietic growth factors

Murine embryonic stem cells are able to differentiate into embryoid bodies (EBs) in vitro in the absence of leukemia-inhibitory factor with the formation of different types of hematopoietic precursors within these EBs. With the aim of determining the in vitro requirements for the continued development of hematopoietic colony-forming cells (CFCs) and their progeny from embryonic stem-derived cells, cells from EBs disrupted after 9 days of formation in the absence of leukemia- inhibitor factor were cultured under different conditions. Low numbers of day-9 EB cells (5 x 10(5) or less) cultured in the presence of several growth factors (interleukin-3 [IL-3], IL-1, c-kit ligand, basic fibroblast growth factor, insulin growth factor-1, IL-6, granulocyte colony-stimulating factor, fetal liver kinase-2 ligand) develop few or no CFCs after 1 week of culture. When these cells are plated on irradiated NIH-3T3 with IL-3 or c-kit ligand or combinations containing these and other growth factors, they are able to generate CFCs for at least 3 weeks. These cultures were found to include granulocytic, monocytic, erythrocytic, and megakaryocytic cells. Transwell cultures in which NIH-3T3 cells were separated from the EB cells and cultures in which cells were replaced by NIH-3T3 conditioned medium showed that the interaction between EB-derived cells and NIH-3T3 is via a soluble factor(s). These studies show that maximal generation of hematopoietic CFCs from precursors present in day-9 EBs is stimulated by a combination of known hematopoietic growth factors and a soluble factor(s) produced by NIH-3T3 cells.     

Variants of 3T3 cells lacking mitogenic response to epidermal growth factor.

We have previously demonstrated that epidermal growth factor (EGF) can serve as a potent mitogen for 3T3 cells. We have now selected variant 3T3 cell lines unable to respond to EGF, in order to define cellular events unique to the EGF response and to distinguish which of these events are necessary and which are merely correlative to mitogenesis. By simultaneously treating cells with EGF and colchicine, we eliminated those cells stimulated by EGF to enter mitosis. Of the eight clonal EGF nonresponder variants selected by this procedure, none retains a functional EGF receptor. The EGF nonresponsive va-riant lines still retain the ability to respond to other mitogens.     

Sodium-stimulated amino acid uptake into isolated membrane vesicles from Balb/c 3T3 cells transformed by simian virus 40.

Mediated uptake of amino acids by membrane vesicles isolated from Balb/c 3T3 cells transformed by simian virus 40 has been demonstrated. Initial rates of transport of radioactively labeled L-leucine and alpha-aminoisobutyric acid were enhanced by the addition of NaCl (100 mM) to the reaction mixture at the start of the uptake process. This enhancement included a prominent "overshoot" during initial uptake. Slight stimulation of alpha-aminoisobutyric acid uptake was seen with K+, but none with Li+. The mediated nature of the uptake event for L-leucine was shown by saturation kinetics and by inhibition with L-valine. The transport assay measured predominantly intravesicular amino acid uptake rather than binding, as shown by the variation of uptake in response to changes in extravesicular osmolarity. Electron microscopy confirmed the presence of closed vesicles. Thus, amino acid transport has been characterized in an in vitro membrane vesicles system which should prove useful for studies of growth control.