Density- and serum-dependent regulation of the Reck tumor suppressor in mouse embryo fibroblasts

Reck is a membrane-anchored glycoprotein identified as a transformation suppressor. Accumulating evidence indicates that Reck negatively regulates a wide spectrum of matrix metalloproteinases and is commonly down-regulated in a variety of malignant solid tumors. Physiological cues that regulate Reck expression, however, remained unknown. In this study, we found that Reck expression was up-regulated at high cell density, low serum, or after treatment with some kinase inhibitors, such as PP2 (Src inhibitor), LY294002 (PI3-kinase inhibitor), and PF573228 (FAK inhibitor), in mouse embryo fibroblasts. Curve fitting indicated that the levels of Reck protein and Reck mRNA are quadratic in the cell density. Other factors, including serum, extracellular matrix components (type I collagen and fibronectin), the kinase inhibitors, and some of their oncogenic targets (v-Src and PIK3CA mutants), modify the shape of the quadratic curve. Comparison of these modifications implicated Src in Reck down-regulation under sparse conditions, PI3-kinase in serum-induced Reck down-regulation, and FAK in Reck down-regulation at high cell density. Fibronectin and type I collagen down-regulated Reck, supporting the role of integrin-FAK signaling in Reck down-regulation at high cell density. Our study has revealed multiple signaling pathways impinging on Reck in cultured mouse embryo fibroblasts and sets a foundation for future studies to find effective Reck inducers of potential value in cancer therapy.     

Effect of concanavalin A and succinyl-concanavalin A on nucleoside and sugar uptake in mouse embryo fibroblasts

We have examined the effect of the tetrameric and dimeric form of Con A at a dose of 50μg ml^?1 on nucleoside and glucose uptake using synchronized mouse embryo fibroblasts undergoing S phase. We have found that thymidine and uridine uptake were depressed by Con A but not significantly by succinyl-Con A. The inhibition was gradual, as it required a suitable time of incubation to become fully manifest and it was of non-competitive type. By contrast the uptake of 2-deoxy glucose was inhibited promptly and to a similar extent by Con A regardless of molecular structure. Kinetic analysis of the modalities of the sugar uptake process indicated an inhibition of competitive type.     

Lactate regulates pyruvate uptake and metabolism in the preimplantation mouse embryo

This study was an investigation of the interaction of lactate on pyruvate and glucose metabolism in the early mouse embryo. Pyruvate uptake and metabolism by mouse embryos were significantly affected by increasing the lactate concentration in the culture medium. In contrast, glucose uptake was not affected by lactate in the culture medium. At the zygote stage, the percentage of pyruvate taken up and oxidized was significantly reduced in the presence of increasing lactate, while at the blastocyst stage, increasing the lactate concentration increased the percentage of pyruvate oxidized. Lactate oxidation was determined to be 3-fold higher (when lactate was present at 20 mM) at the blastocyst stage compared to the zygote. Analysis of the kinetics of lactate dehydrogenase (LDH) determined that while the V(max) of LDH was higher at the zygote stage, the K(m) of LDH was identical for both stages of development, confirming that the LDH isozyme was the same. Furthermore, the activity of LDH isolated from both stages was reduced by 40% in the presence of 20 mM lactate. The observed differences in lactate metabolism between the zygote and blastocyst must therefore be attributed to in situ regulation of LDH. Activity of isolated LDH was found to be affected by nicotinamide adenine dinucleotide(+) (NAD(+)) concentration. In the presence of increasing concentrations of lactate, zygotes exhibited an increase in autofluorescence consistent with a depletion of NAD(+) in the cytosol. No increase was observed for later-stage embryos. Therefore it is proposed that the differences in pyruvate and lactate metabolism at the different stages of development are due to differences in the in situ regulation of LDH by cytosolic redox potential.     

Decreased rate of uridine and glycerin uptake in L cells resistant to ethidium bromide

A comparison was made among rates of uptake of 3H-uridine, 3H-glycerol and 3H-D-xylose into mouse fibroblasts of line L sensitive to ethidium bromide (EB), and into EB-resistant cells obtained from this line by selection. Constants of uridine transport and phosphorylation were determined. For EB sensitive L cells Kt was 162 +/- 27 microM, Vt was 7.5 +/- 0.7 microM/sec. For EB resistant cells Kt was 178 +/- 27 microM, and Vt was 4.6 +/- 0.2 microM/sec. Thus, the transport rate of uridine in resistant cells was twice lower than in EB sensitive cells. The rate of uridine phosphorilation in EB resistant cells was by three times lower than in EB sensitive ones. The uptake of glycerol into resistant cells was also lowered. There was no difference in transport of 3H-D-xylose between sensitive and resistant cells. The data obtained may suggest some changes in plasma membrane in the EB resistant cells.     

Genetic dissection of nodal function in patterning the mouse embryo

Loss-of-function analysis has shown that the transforming growth factor-like signaling molecule nodal is essential for mouse mesoderm development. However, definitive proof of nodal function in other developmental processes in the mouse embryo has been lacking because the null mutation blocks gastrulation. We describe the generation and analysis of a hypomorphic nodal allele. Mouse embryos heterozygous for the hypomorphic allele and a null allele undergo gastrulation but then display abnormalities that fall into three distinct mutant phenotypic classes, which may result from expression levels falling below critical thresholds in one or more domains of nodal expression. Our analysis of each of these classes provides conclusive evidence for nodal-mediated regulation of several developmental processes in the mouse embryo, beyond its role in mesoderm formation. We find that nodal signaling is required for correct positioning of the anteroposterior axis, normal anterior and midline patterning, and the left-right asymmetric development of the heart, vasculature, lungs and stomach.     

Regulation of uridine uptake by serum and insulin in density-inhibited 3T3 cells

Stimulation of nucleoside uptake in quiescent 3T3 cells by insulin and serum is preceded by a substantial lag phase. Our findings point to the length of the lag phase as a major target for regulation. The length of such phase varies markedly with the concentration of insulin (10^?9-10^?6 M) or serum (0.5–10%) but it is not eliminated by high, saturating levels of the activating agents. Further, variations in the temperature at which the stimulation process occurs 24–39°C), addition of compounds like prostaglandin E₁ (1–5 μg/ml) or theophylline (0.4 mM) and differences in the age of the cultures primarily affect the length of the lag time while the final uptake rates achieved are remarkably constant. Analysis of the temporal order of the events in the lag phase reveals that there is a discrete temperature-sensitive period located in the early and middle part of the lag, while the prostaglandin E₁-sensitive step(s) appear to be toward the end of the lag. The transition from the basal to the stimulated rate of uptake is abrupt. Indeed, the kinetics of activation does not fit a simple exponential law but a high power of an exponential, suggesting that the switching mechanism involves cooperative steps. Since the transition is abrupt, the nucleoside uptake system exists largely in two alternative states either switched off or on. The regulation of the lag period is by the control of the time at which this switch occurs. On the basis of the data presented here, we propose a working hypothesis of uptake stimulation.     

Primordial germ cells in the mouse embryo during gastrulation

With the aid of a whole-mount technique, we have detected a small cluster of alkaline phosphatase (ALP)-positive cells in whole mounts of mid-primitive-streak-stage embryos, 7-7 1/4 days post coitum (dpc). Within the cluster, about 8 cells contain a small cytoplasmic spot, intensely stained for ALP activity and possibly associated with an active Golgi complex. The cluster lies just posterior to the definitive primitive streak in the extraembryonic mesoderm, separated from the embryo by the amniotic fold. Towards the end of gastrulation, the number of cells containing the ALP-positive spot rises to between 50 and 80. Thereafter the number of cells in the extraembryonic cluster declines, and similar cells start to be seen in the mesoderm of the primitive streak and then in the endoderm. At 8 dpc, about 125 ALP-stained cells are found, mainly in the hindgut endoderm and also at the base of the allantois, their appearance and location at this stage agreeing closely with previous reports on primordial germ cells (PGCs). Embryos from which the cluster area has been removed at the 7-day stage are devoid of PGCs after culture for 48 h, whereas the excised tissue is rich in PGCs. We argue that the cells in the cluster are indeed primordial germ cells, at a stage significantly earlier than any reported previously. This would indicate that the PGC lineage in the mouse is set aside at least as early as 7 dpc, possibly as one of the first 'mesodermal' cell types to emerge, and that its differentiation, as expressed by ALP activity, is gradual.     

Serum-free mouse embryo cells: growth responses in vitro

We have derived serum-free mouse embryo (SFME) cultures in a basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and fibronectin. These cells are nontumorigenic, lack gross chromosomal aberrations, and exhibit several other unique properties, including dependence on EGF for survival and growth inhibition by serum. We have examined the concentration dependence of the growth stimulatory effects of protein supplements used in the SFME medium formulation and surveyed other supplements that might act as alternative or complementary additions to the culture medium. Insulin could be replaced by insulin-like growth factor I and EGF could be replaced by transforming growth factor alpha in the same concentration range. Transferrin could be replaced by higher concentrations of lactoferrin. Deterioration of cultures in the absence of EGF began within 8 hours of the removal of the growth factor, and could be prevented by the addition of fibroblast growth factor/heparin-binding growth factor. Attachment proteins other than fibronectin were effective on SFME cells, but limited success was obtained when substituting other lipid preparations for HDL. These data introduce a precise system for exploring the unusual characteristics of SFME cells and contribute additional information that may be useful in the extension of these approaches to other cell types and species.     

Alterations of mouse embryo cells during in vitro aging

Trichocysts from the ciliated protozoan Paramecium aurelia have been solubilized in aqueous solution at neutral pH by heating to temperatures of 70 °C for 10 min. The product of such solubilization appears identical to that achieved by the previous method using trichocysts solubilized in sodium dodecyl sulfate (SDS) when examined by SDS polyacrylamide disc gel electrophoresis. While the solubilization of the trichocyst is virtually total in distilled water, no more than 20% of the total protein solubilizes in the presence of salts even in amounts as low as 0.05 M. Heat solubilized trichocysts show two principal components when run on standard 10% polyacrylamide disc gels and two bands when run on SDS polyacrylamide disc gels. The relationship between the two bands seen on standard gels and the two bands seen on SDS gels is unknown at this time. These results represent the first successful solubilization of this organelle in aqueous solution at neutral pH as opposed to previous reports requiring strong denaturants such as SDS, guanidine hydrochloride, and pH extremes. While the solubilization of trichocysts by heat most probably results in denaturation of the constituent proteins, it does occur in aqueous solution without the use of strong denaturants or pH extremes allowing the use of standard analytical procedures not possible in the presence of these reagents.     

Divalent antibodies to mouse embryonal carcinoma cells inhibit compaction in the mouse embryo

Divalent antibodies from two antisera to embryonal carcinoma (EC) cells, F9 line, inhibited compaction in the preimplantation mouse embryo. One of these antisera, AF9-2, completely inhibited compaction at the 8-cell stage when the embryos were continuously incubated in a $\text{1}\text{100}$ dilution of the antiserum in culture medium from the 4-cell stage. Cell divisions continued and no cellular degeneration occurred. When cultured control embryos (preimmune and nonimmune sera) were normal blastocysts, many of the AF9-2-treated embryos were characterized by vacuolated cells and rounded rather than squamous, trophoectodermal cells. Anti-mouse spleen serum ($\text{1}\text{10}$, $\text{1}\text{100}$) had no effect on development. Purified divalent IgGs from AF9-2 (ammonium sulfate precipitation and DEAE chromatography) also were inhibitory at 30 μg/ml. Inhibition of compaction by AF9-2 was reversible. When uncompacted midmorula-stage embryos in AF9-2 ($\text{1}\text{10}$) were transferred to medium without serum, there was a threefold increase in the percentage (70%) of normal blastocysts at the end of culture. Fluorescence microscopy demonstrated that AF9-2 antibodies, unlike preimmune and nonimmune sera, were bound to the surface of 8-cell and early morula-stage embryos. Inhibition by AF9-2 does not occur merely by nonspecifically coating cell surfaces so that they no longer recognize each other, since antispleen antibodies show similar binding by immunofluorescence but no inhibition. This study provides strong evidence that AF9-2 has specific divalent antibodies which block morphogenesis. Since newly compacted embryos lost their compacted appearance in AF9-2, these divalent antibodies cause a loss of cell adhesion, do not extensively cross-link adjacent cell surfaces, and cannot cause the compacted phenotype by agglutination.