Morphological and proliferative characteristics of human breast tumor cells cultured on plastic and in collagen matrix

Summary Collagen, a major component of the extracellular matrix, is important in maintaining the in vivo characteristics of epidermal cells in vitro. In the present study, the morphological and proliferative characteristics of two human mammary epithelial cell lines (T-47D and MCF-7) cultured in cowhide collagen (Vitrogen 100) were studied. When grown in collagen, the tumor cells displayed a spherical shape and formed multilayered, tumorlike aggregates; desmosomes were observed between cells. In contrast, both cell lines grew as monolayers on plastic substratum; cells were characteristically flat and polygonal. When grown in collagen matrix, the human breast cancer cells became more dependent on serum for growth: cells proliferated in the presence of 10% fetal bovine serum (FBS) but failed to grow in 1% serum. On the other hand, these cells proliferated rapidly in 1% serum when they were grown on plastic. Even in 10% serum the doubling time of cells cultured in collagen was longer than that of cells maintained on plastic. In addition, cells cultured in collagen proliferated rapidly in a serum-free medium containing insulin, epidermal growth factor (EGF), estrogen, and transferrin. The collagen gel system may be useful for characterizing physiologically important trophic factors that regulate the proliferation and other functions of human breast tumor cells. The advice of Drs. J. A. Paterson and B. Dronzek in the electron microscopy studies is appreciated. This research was supported by the Medical Research Council of Canada. Clement K. H. Leung was supported by a University of Manitoba graduate fellowship. Portions of this work were reported at the Twentieth Annual Meeting of the American Society for Cell Biology held in Cincinnati, Ohio, November 14–18, 1980.     

Barbiturate Attenuation of Brain Free Fatty Acid Liberation During Global Ischemia

Abstract: To find a biochemical basis for the increased tolerance of the brain to anoxia during barbiturate anesthesia, we studied whole-brain free fatty acids (FFA) at various times after decapitation of awake and pentobarbital-anesthetized rats. Post-decapitation, the brains were kept at 37°C for 1 to 60 min before freezing in liquid N₂. Nonischemic brains were frozen in liquid N₂, using a rapid sampling technique. Whole-brain arachidonic, stearic, oleic, linoleic, and palmitic acids were quantitated by gas-liquid chromatography. In unanesthetized, nonischemic brain, total FFA was 1226 ± 121 nmol/g brain ( n = 12) and was unaffected by pentobarbital anesthesia (1126 ± 86 nmol/g brain, n = 11), except for a reduction in arachidonic acid. Total FFA in unanesthetized and pentobarbital-anesthetized rats transiently declined between 0 and 1 min of ischemia, and then rose linearly for up to 60 min, with consistently lower values in pentobarbital-treated rats, the greatest attenuation being that of arachidonic and stearic acid liberation. Brain FFA liberation during global ischemia is the first known biochemical variable directly correlated with the duration (i.e., severity) of global ischemia. The attenuation of brain FFA liberation and especially of arachidonic and stearic acids may be the biochemical basis of barbiturate attenuation of ischemic brain injury.     

Cross-linking of G-proteins to the prolactin receptor in rat NB2 lymphoma cells

In Nb2 cell membranes, two guanine nucleotide-binding protein (G-protein) species (Mr 43.5 and 46.5 kD) were [32P]-ADP-ribosylated by cholera toxin, while a single protein (Mr 41.5 kD) was [32P]-ADP-ribosylated by pertussis toxin. Immunostaining indicated two immunoreactive prolactin (PRL) receptor moieties of 56 and 64 kD. Cross-linking with ethylene glyco bis[succinimidyl-succinate] (mol. length of 16.1 A) generated a high mol. wt., [32P]-ADP-ribosylated band of 140-160 kD which also showed immunoreactivity with antiserum to the PRL receptor. Other cross-linkers with shorter molecular lengths (8.6 - 11.4 A) were ineffective. These findings indicate that the Nb2 lactogen receptor is complexed with G-proteins and provide evidence for the role of G-proteins in mediating PRL-stimulated mitognesis in Nb2 cells.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9¹⁴-C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5–3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

Rho mediates the shear-enhancement of endothelial cell migration and traction force generation

The migration of vascular endothelial cells in vivo occurs in a fluid dynamic environment due to blood flow, but the role of hemodynamic forces in cell migration is not yet completely understood. Here we investigated the effect of shear stress, the frictional drag of blood flowing over the cell surface, on the migration speed of individual endothelial cells on fibronectin-coated surfaces, as well as the biochemical and biophysical bases underlying this shear effect. Under static conditions, cell migration speed had a bell-shaped relationship with fibronectin concentration. Shear stress significantly increased the migration speed at all fibronectin concentrations tested and shifted the bell-shaped curve upwards. Shear stress also induced the activation of Rho GTPase and increased the traction force exerted by endothelial cells on the underlying substrate, both at the leading edge and the rear, suggesting that shear stress enhances both the frontal forward-pulling force and tail retraction. The inhibition of a Rho-associated kinase, p160ROCK, decreased the traction force and migration speed under both static and shear conditions and eliminated the shear-enhancement of migration speed. Our results indicate that shear stress enhances the migration speed of endothelial cells by modulating the biophysical force of tractions through the biochemical pathway of Rho-p160ROCK.     

THE BIOLOGICAL ACT'IVITIES OF YELLOW AZALEA,RHODODENDRON MOLLE G. DON AGAINST THE VEGETABLE LEAFMINER,LIRIOMYZA SATIVAE (DIPTERA: AGROMYZIDAE) *

Abstract The results of laboratory and greenhouse bioassays indicated that Rhodojaponin‐ III (Abbr. R‐ 1) and extracts of flowers from Rhododendron molle G. Don possessed signficant feeding inhibition and insectcidal properties against the larvae and adults of Liromyzia sativae. Treated with 500 mg/L R‐ III 1 000 mg/L molosul‐tap, and 10 000 mg/L methanol(MeOH) ethyl acetate (EtOAc), CH₂Cl₂, methanol‐water (MeOH‐H₂O) extracts the rates of feeding inhibition were 77. 34 % 74.30 % 82.15 % 77.50 % 67. 33 % 62.85 % against the 2nd instar larvae, and were 67.66% 55.21 % 49.72% 54.26% 46.81 % 38.53% against the 3rd instar larvae, respectively;LC₅₀ values against the 2nd instar larvae were 208.65, 166.05, 2.74 ± 10³,766.72, 5.95 ± 10³, 1.85 ± 10³mg/L, and against 3rd larvae were 300.62, 256.00, 4.33 ±10³, 1.03 ± 10³,9.79 ± 10³, and 2.62± 10³mg/L, respectively. Against the adults, LC₅₀ values of R‐III EtOAc extract and molosultap were 159.07.723.87 and 134.55mgL respectively after treatment for 24 h.     

Properties and purification of a glucose-regulated protein from chick embryo fibroblasts.

A glucose-regulated protein of molecular weight 78,000 (GRP-78) had been purified from a membrane fraction isolated from viral transformed chick embryo fibroblasts. Purification was achieved by extraction of the membrane fraction with Triton X-100, and chromatography on diethylaminoethyl-cellulose and hydroxyapatite. The purified protein exhibited one single spot on two-dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has a pI of about 5.3. A monospecific antiserum to GRP-78 was generated in a goat. Immunofluorescence studies using affinity purified antibodies to GRP-78 revealed that this protein was not exposed on the cell surface but was localized in a granular vesicular network inside the cell that resembles the distribution of endoplasmic reticulum. The availability of purified GRP-78 and a specific antiserum to it should prove useful in elucidating the role of this protein in glucose metabolism and its relationship to malignant transformation.     

Dynamics of Posttranslational Modification Systems: Recent Progress and Future Directions

Posttranslational modification of proteins is important for signal transduction, and hence significant effort has gone toward understanding how posttranslational modification networks process information. This involves, on the theory side, analyzing the dynamical systems arising from such networks. Which networks are, for instance, bistable? Which networks admit sustained oscillations? Which parameter values enable such behaviors? In this Biophysical Perspective, we highlight recent progress in this area and point out some important future directions. Along the way, we summarize several techniques for analyzing general networks, such as eliminating variables to obtain steady-state parameterizations, and harnessing results on how incorporating intermediates affects dynamics.