Glucocorticoid effects on membrane lipid mobility during differentiation of murine B lymphocytes

The lateral motion of membrane lipids on lipopolysaccharide-stimulated murine B lymphocytes was measured using photobleaching recovery techniques. The mobility of the phospholipid analog 3,3'-dioctadecylindocarbocyanine iodide (DiI) was measured at 37 degrees C on B lymphocytes 48 h after stimulation by various concentrations of lipopolysaccharide. DiI mobility on lymphoblasts from cultures stimulated with 10 micrograms/ml lipopolysaccharide was reduced 50% compared with unstimulated, small B cells. However, both lower and higher lipopolysaccharide concentrations caused some decrease in lipid mobility. Lipid mobility was measured on B cells stimulated with 10 micrograms/ml lipopolysaccharide at zero time, on lymphoblasts at 18, 24, 48 and 72 h, and on immunoglobulin (Ig) -secreting lymphocytes at 96 h. The diffusion coefficient of DiI on both control and lipopolysaccharide-treated cells at zero time is 6.3 X 10(-9) cm2 X s-1. This value remains unchanged for unstimulated cells over 72 h. Lipid mobility of lipopolysaccharide-activated lymphoblasts decreased during incubation with lipopolysaccharide to 5.0, 3.4, 2.8 and 2.4 X 10(-9) cm2 X s-1 after 18, 24, 48 and 72 h, respectively. DiI mobility on immunoglobulin (Ig) -secreting lymphocytes identified at the foci of Protein A-coated sheep red blood cells plaques is 8.6 X 10(-9) cm2 X s-1, a value similar to that of unstimulated B cells. The effect of introducing various concentrations of a synthetic glucocorticoid, triamcinolone acetonide (TA), to 48 h lipopolysaccharide-stimulated cells for 6 h was examined. Maximal TA effect was observed at a concentration of 10(-7) M, which caused an increase in lipid mobility to 7.5 X 10(-9) cm2 X s-1. Exposing resting B cells (t = 0) or lymphoblasts (t = 24, 48 or 72 h) to TA for 3 h had no effect on lipid mobility. Treatment for 6 h with 10(-7) MTA increased DiI diffusion to 12.6, 9.9, 7.5 and 6.8 X 10(-9) cm2 X s-1 on control cells and on 24, 48 and 72 h lipopolysaccharide-activated lymphoblasts, respectively. A longer incubation of 12 h with 10(-7) MTA caused no further change in lipid lateral diffusion. The response was glucocorticoid-specific. In lymphoblasts (48 h) incubated an additional 6 h with 10(-7) MTA and a 100-fold excess of cortexolone or progesterone, the increase in lipid mobility was substantively blocked; estradiol and testosterone had no effect on lipid lateral diffusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lateral mobility of a lipid analog in the membrane of irreversible sickle erythrocytes

The major feature of sickle cell anemia is the tendency of erythrocytes to sickle when exposed to decreased oxygen tension and to unsickle when reoxygenated. Irreversible sickle cells (ISCs) are sickle erythrocytes which retain bipolar elongated shapes despite reoxygenation. ISCs are believed to owe their biophysical abnormalities to acquired membrane alterations which decrease membrane deformability. While increased membrane surface viscosity has been measured in ISCs, the lateral dynamics of membrane lipids in these cells have not heretofore been examined. We have measured the lateral diffusion of the lipid analog 3,3'-dioctadecylindocyanine iodide (DiI) in the plasma membrane of intact normal erythrocytes, reversible sickle cells (RSCs), and irreversible sickle cells by fluorescence photobleaching recovery (FPR). The diffusion coefficients +/- standard errors of the mean of DiI in intact normal red blood cells (RBCs), RSCs, and ISCs at 37 degrees C are (8.06 +/- 0.29) X 10(-9) cm2 X s-1, (7.74 +/- 0.22) X 10(-9) cm2 X s-1, and (7.29 +/- 0.24) X 10(-9) cm2 X s-1, respectively. A similar decrease in the diffusion coefficient of DiI in the plasma membranes of the three cell types was observed at 4, 10, 17, 23, and 30 degrees C. ANOVA analysis of the changes in DiI diffusion showed significant differences between the RBC and ISC membranes at all temperatures examined. The characteristic breaks in Arrhenius plots of the diffusion coefficients for the RBCs, RSCs, and ISCs occurred at 20, 19, and 18.6 degrees C, respectively. Photobleaching recovery data were used to estimate (Boullier, J.A., Melnykovich, G. and Barisas, B.G. (1982) Biochim. Biophys. Acta 692, 278-286) the microviscosities of the plasma membranes of the three cell types at 25 degrees C. We find significant differences between our microviscosity values and those obtained in previous fluorescence depolarization studies. However, both methods indicate qualitatively similar differences in membrane microviscosity among the various cell types.     

Dissociation of inositol trisphosphate from diacylglycerol production in Rous sarcoma virus-transformed fibroblasts

The metabolism of phosphatidylinositol (PI) and related intermediates was studied in uninfected and Rous sarcoma virus-(RSV) infected chicken embryo fibroblasts (CEFs). Cells infected with wild-type RSV exhibited twofold increases in steady-state concentrations of inositol trisphosphate (IP3) and inositol bisphosphate (IP2) as compared to uninfected CEFs. In addition, increased concentrations of IP3 and IP2 were observed in CEFs infected with the RSV temperature-sensitive transformation mutant NY72-4 when maintained at the permissive temperature (35 degrees C) for greater than 24 h. Slight increases were observed in the amounts of inositol lipids in RSV-transformed cells. Phosphoinositol metabolic changes were related to transformation and not to viral infection since CEFs infected with NY72-4, maintained at the nonpermissive temperature (41.5 degrees C), revealed amounts of phosphoinositols similar to that of uninfected cells. CEFs infected with a transformation-defective virus exhibited PI metabolic changes intermediate between those of transformed and nontransformed cells. NY72-4 CEF exhibited no increase in phosphoinositol concentrations before 8 h incubation at 35 degrees C, indicating that the transformation-specific changes in inositol metabolism were a delayed event. Furthermore, inositol turnover was not activated during this time. In contrast to the case of inositol metabolism, significant increases in diacylglycerol (DAG) concentrations were observed within 15-30 min after shift of NY72-4 CEFs to 35 degrees C. These findings suggest that (a) the major changes in inositol metabolism are specific for RSV-transformed cells; (b) transformation-specific changes in phosphoinositol content in RSV-infected CEFs are not an early effect of the expression of pp60v-src; and (c) increases in the DAG content of transformed cells occur before changes in inositol metabolism, indicating that DAG may be derived from other lipid sources.     

Lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in the membrane of differentiating HL-60 and U-937 cells assessed with fluorescence recovery after photobleaching (FRAP)

The promyelocytic leukemia cell line HL-60 and the histiocytic cell line U-937 were grown in suspension culture. They were induced to differentiate during 5-d cultivation in the presence of dimethylsulfoxide (DMSO; 1.3% w/v) or phorbol-12-myristate-acetate (PMA; 10(-7) M), which yields granulocyte- and macrophage-like cells, respectively. Differentiation was evidenced by increased capacity to recognize and phagocytize IgG- or complement-coated yeast particles. Aliquots taken from the cultures with and without DMSO (or PMA) were spun down directly on glass microscope slides, washed, labeled with fluoresceinated wheat germ agglutinin (WGA), and directly examined at room temperature for the rate of fluorescence recovery after photobleaching (FRAP). It was found that cultivation of the HL-60 and the U-937 cells in the presence of DMSO, which yields granulocyte-like cells, reduced the average value of lateral diffusion coefficient D (X 10(10] from 1.72 +/- 0.13 cm2s-1 to 0.97 +/- 0.13 cm2s-1 and from 1.77 +/- 0.11 cm2s-1 to 0.82 +/- 0.13 cm2s-1, respectively. U-937 cells grown with PMA also showed a reduction of D(X 10(10] to 0.88 +/- 0.10 cm2s-1. There was a larger immobile fraction of fluorescence in the HL-60 cells than in the U-937 cells, viz., 70-80% compared to 10-50%. The total number of binding sites for WGA was not altered, but the surface density changed, since the HL-60 and the U-937 cells became smaller and larger, respectively, when grown in the presence of DMSO. It is concluded that differentiation reduces the average lateral mobility of the WGA-binding membrane component by a factor around 2.     

High lateral mobility of endogenous and transfected alkaline phosphatase: a phosphatidylinositol-anchored membrane protein

The lateral mobility of alkaline phosphatase (AP) in the plasma membrane of osteoblastic and nonosteoblastic cells was estimated by fluorescence redistribution after photobleaching in embryonic and in tumor cells, in cells that express AP naturally, and in cells transfected with an expression vector containing AP cDNA. The diffusion coefficient (D) and the mobile fraction, estimated from the percent recovery (%R), were found to be cell-type dependent ranging from (0.58 +/- 0.16) X 10(-9) cm2s-1 and 73.3 +/- 10.5 in rat osteosarcoma cells ROS 17/2.8 to (1.77 +/- 0.51) X 10(-9) cm2s-1 and 82.8 +/- 2.5 in rat osteosarcoma cells UMR106. Similar values of D greater than or equal to 10(-9) cm2s-1 with approximately 80% recovery were also found in fetal rat calvaria cells, transfected skin fibroblasts, and transfected AP-negative osteosarcoma cells ROS 25/1. These values of D are many times greater than "typical" values for membrane proteins, coming close to those of membrane lipid in fetal rat calvaria and ROS 17/2.8 cells (D = [4(-5)] X 10(-9) cm2s-1 with 75-80% recovery), estimated with the hexadecanoyl aminofluorescein probe. In all cell types, phosphatidylinositol (PI)-specific phospholipase C released 60-90% of native and transfection-expressed AP, demonstrating that, as in other tissue types, AP in these cells is anchored in the membrane via a linkage to PI. These results indicate that the transfected cells used in this study possess the machinery for AP insertion into the membrane and its binding to PI. The fast AP mobility appears to be an intrinsic property of the way the protein is anchored in the membrane, a conclusion with general implications for the understanding of the slow diffusion of other membrane proteins.     

Transformation of rat cells by fusion-infection with Rous sarcoma virus

The transformation of a rat cell line, 3Y1, by nonmammalian tropic strains of avian sarcoma virus was tested using cell-virus fusion mediated by Sendai virus or polyethylene glycol. Furthermore, the establishment of several transformed 3Y1 cell clones induced by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), its derivative mutants, and the Bryan high-titer strain of RSV is reported. The presence and expression of the viral genomes in these cells were examined, and all transformed cell clones tested were found to contain rescuable RSV genomes when they had been fused with normal chicken embryo fibroblast cells or those preinfected with Rous-associated virus type 1. However, the gag gene product, pr76, was barely detectable in wild-type RSV-transformed cells, whereas it was produced in considerable amounts in cells transformed by env-deleted mutants, the Bryan high-titer strain of RSV and NY8 derived from the Schmidt-Ruppin strain of RSV.     

Lipid diffusibility in the intact erythrocyte membrane

The lateral diffusion of fluorescent lipid analogues in the plasma membrane of intact erythrocytes from man, mouse, rabbit, and frog has been measured by fluorescence photobleaching recovery (FPR). Intact cells from dystrophic, normoblastic, hemolytic, and spherocytotic mouse mutants; from hypercholesterolemic rabbits and humans; and from prenatal, neonatal, and juvenile mice have been compared with corresponding normals. The lateral diffusion coefficient (D) for 3,3'-dioctadecylindodicarbocyanine iodide (DiI[5]) in intact normal human erythrocytes is D = 8.2 +/- 1.2 X 10(-9) cm2/s at 25 degrees C and D = 2.1 +/- 0.4 X 10(-8) cm2/s at 37 degrees C, and varies approximately 50-fold between 1 degree and 42 degrees C. The diffusion constants of lipid analogue rhodamine-B phosphatidylethanolamine (RBPE) are about twice those of DiI[5]. The temperature dependence and magnitude of D vary by up to a factor of 3 between species and are only influenced by donor age in prenatals. DiI[5] diffusibility is not perturbed by the presence of calcium or local anesthetics or by spectrin depletion (via mutation). However, lipid-analogue diffusibility in erythrocyte ghosts may differ from intact cells. Dietary hypercholesterolemia in rabbits reduces the diffusion coefficient and eliminates the characteristic break in Arrhenius plots of D found in all other cells studied except frog.     

The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity

Tyrosine-specific phosphorylation of cellular proteins has been implicated in the neoplastic transformation of cells by Rous sarcoma virus (RSV). One of the putative substrates for the src gene product (pp60v-src) of RSV is the cytoskeletal protein vinculin, giving rise to the hypothesis that tyrosine-specific phosphorylation of vinculin disrupts adhesion plaque integrity, leading to the characteristic rounded morphology of RSV-transformed cells. We have investigated this hypothesis by analysing the properties of fibroblasts transformed by conditional and non-conditional mutants of RSV which confer different morphologies on infected cells, with respect to formation of microfilament bundles, formation of vinculin-containing adhesion plaques, the deposition of a fibronectin-containing extracellular matrix, the localization of pp60v-src and the tyrosine-specific phosphorylation of vinculin. Cells transformed by the temperature-sensitive (ts) RSV mutant LA32 cultured at 41 degrees C were morphologically normal, and contained prominent microfilament bundles and well-developed adhesion plaques. However, these cells had a fully active pp60v-src kinase, had pp60v-src concentrated in their adhesion plaques and contained vinculin which was heavily phosphorylated on tyrosine residues. Cells transformed by a recovered avian sarcoma virus, rASV 2234.3 exhibited a markedly fusiform morphology with pp60v-src concentrated in well-developed adhesion plaques and an elevation of the phosphotyrosine content of vinculin. Cells transformed by LA32 at restrictive temperature comprise morphologically normal cells, indistinguishable from untransformed CEF, yet which contain tyrosine-phosphorylated vinculin and suggest that neither tyrosine-specific phosphorylation of vinculin nor pp60v-src concentration in adhesion plaques is sufficient for the rounded morphology of RSV-transformed cells.     

Early release of the density-dependent inhibition of phosphate uptake and ATP synthesis after src gene expression in chick embryo fibroblasts

Our results showed that the expression of the src gene in chick embryo fibroblasts (CEF) released the density-dependent inhibition (DDI) of phosphate metabolism (phosphate uptake and phosphorylation of small organic compounds). With increasing cell density, phosphate metabolism decreased by 58% in normal CEF and, in contrast, increased by 20% in Rous sarcoma virus (RSV)-transformed CEF. The same change in the DDI was observed in CEF infected by NY68 (a ts mutant for transformation of RSV) and maintained at the permissive temperature (37 degrees C) instead of the restrictive temperature (41.5 degrees C) for the expression of transformation. An interesting feature was that the release of the DDI of phosphate metabolism was an early event in the process of transformation, since it was almost concomitant with the stimulation of the pp60 src kinase activity following the shift from 41.5 to 37 degrees C of NY68 CEF. The phosphorylation of small organic compounds (Po) was more strongly increased by the change in temperature than was 32Pi accumulation. Furthermore, the percentage increases of Po and adenosine triphosphate (ATP) labelling with 32P were similar, suggesting that the expression of src gene enhanced ATP synthesis. In glucose-free medium, the stimulation of Po-labelling was still observed but was decreased. Therefore the activation of glycolytic activity is not an absolute requirement, but is necessary for the maximum effect of transformation on the release of DDI of phosphate metabolism. Oligomycin added in complete medium did not prevent the increase in Po-labelling. From these results, we assumed that ATP turnover was stimulated as a consequence of enhanced ATP degradation. We verified that the stimulation of Po phosphorylation was not a consequence of increased ATP utilization for RNA or protein synthesis. The stimulation of Po labelling was specifically abolished by quercetin. This drug inhibited the transformed cells more strongly than the non-transformed cells.     

78-kilodalton glucose-regulated protein is induced in Rous sarcoma virus-transformed cells independently of glucose deprivation.

To identify mRNAs with altered expression in Rous sarcoma virus (RSV)-transformed cells, we screened a chicken embryo fibroblast (CEF) cDNA library by differential hybridization. One clone, designated R1H, showed markedly elevated mRNA expression in RSV-transformed cells. Nucleotide sequence analysis indicated that R1H mRNA encodes 78-kilodalton glucose-regulated protein (GRP78). Chicken GRP78 was found to be very highly conserved in comparison with rat GRP78 (96% identity between chicken and rat amino acid sequences). In contrast to previous observations, we found that GRP78 was induced in RSV-transformed cells in the absence of glucose deprivation. When cells were grown in glucose-supplemented medium, the level of GRP78 mRNA was approximately fivefold higher in RSV-transformed CEF than in transformation-defective virus-infected or uninfected CEF. Similar changes in GRP78 protein content were also found. Using a temperature-sensitive mutant of RSV and supplemental glucose, we found a gradual increase in the level of GRP78 mRNA beginning at 4 h after shiftdown to permissive temperature. Uridine supplementation did not block the induction seen in CEF infected with a temperature-sensitive mutant. These results indicate that GRP78 is induced by p60v-src in the absence of glucose deprivation.     

Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility.

We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with eosin maleimide and fluorescein thiosemicarbazide, respectively. The band 3 diffusion rate increased from 1.7 x 10(-11) cm2 s-1 to 6.0 x 10(-11) cm2 s-1 as oxygen tension was decreased from 156 to 2 torr, and a further increase to 17 x 10(-11) cm2 s-1 occurred as oxygen tension was decreased from 2 to 0 torr. The fractional mobility of band 3 decreased from 58 to 32% as oxygen tension was decreased from 156 to 0 torr. The phospholipid diffusion coefficient remained constant as oxygen tension was decreased from 156 to 20 torr, but increased from 2.3 x 10(-9) cm2 s-1 to 7.1 x 10(-9) cm2 s-1 as oxygen tension was decreased from 20 to 0 torr. Neither the diffusion coefficient nor the fractional mobility of glycophorins changed significantly at low oxygen tension. Under non-bleaching excitation conditions, intensities of fluorescence emission were identical for oxygenated and deoxygenated eosin-labeled RBCs. Deoxygenated eosin-labeled RBCs required 160-fold greater laser intensities than did oxygenated RBCs to achieve comparable extents of photobleaching, however. Oxygen seems to act as a facilitator of fluorophore photobleaching and may thereby protect the fluorescently labeled red cell membrane from photodamage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral diffusion of the secretory component (SC) in the basolateral membrane of the human colon carcinoma cell line HT29 assessed with fluorescence recovery after photobleaching

The lateral diffusion of the secretory component (SC), acting as a receptor for dimeric IgA in the basolateral side of intestinal epithelial cells, was studied in the human colonic carcinoma cell line HT29. The HT29 cells were grown in Dulbecco's modified Eagle's medium in which galactose had been substituted for glucose to promote development of small intestine-like cells, with a distinct separation of the basolateral side from the apical surface. The SC was stained with rhodamine-labeled polyclonal anti-human SC rabbit antibodies (Ig) or Fab fragments, and the lateral mobility was assessed with the fluorescence recovery after photobleaching technique. The average lateral diffusion was consistent with a diffusion constant of 7.7 +/- 2.0 (mean value +/- SD; n = 29) and 7.1 +/- 2.3 (n = 30) x 10(-10) cm2s-1 for Ig-and Fab-labeled receptors, respectively, which is slower than lipid diffusion but is similar to that found for other membrane receptors. The corresponding values for the fraction of mobile receptors were 66 +/- 13% and 71 +/- 12%, respectively. Cells were labeled from the top of the culture plate, and cells adjacent to a mechanically made rift or a natural opening in the cell monolayer were labeled more strongly, confirming the microscope-based impression that the basolateral surface primarily harboured the SC receptor.     

Neuraminidase activity and cell surface sialic acid turnover in Rous sarcoma virus transformed chick embryo fibroblasts

Neuraminidase activity of Rous sarcoma virus transformed chick embryo fibroblasts (RSV-CEF) was assayed using an exogenous substrate, neuraminlactitol-[3H], and endogenous, cell surface [14C]-N]-acetyl-neuraminic acid. RSV-CEF had higher neuraminidase activity toward both substrates than did chick embryo fibroblasts (CEF) or nontransformed, Rous associated virus infected CEF (RAV-CEF). The total sialic acid content of RSV-CEF was lower than CEF or RAV-CEF, and more of the total sialic acid was accessible to extracellular Clostridium perfringens neuraminidase. Activity of the enzymes synthesizing and degrading the substrate for sialyltransferase, cytidine-5'-monophosphate-N-acetyl-neuraminic acid (CMP-AcNeu) was measured in order to determine whether control of substrate levels for sialyltransferase might contribute to the decreased levels of glycoprotein bound sialic acid. No change in activity of these enzymes was found in RSV-CEF as compared to CEF or RAV-CEF.     

Lateral mobility of plasma membrane lipids in normal and transformed keratinocytes

In this study we have examined possible differentiation-dependent modulations in plasma membrane lipid properties in normal keratinocytes, SV-40 transformed keratinocytes (SVK14) and a number of squamous carcinoma (SCC) cells. In normal keratinocytes the lateral diffusion coefficient of plasma membrane lipids (D) differs significantly for cells cultured permanently under low and normal Ca2+-conditions (5.16 x 10(-9) and 3.27 x 10(-9) cm2/s, respectively). When differentiation is induced by exposing low Ca2+-cultured cells to normal Ca2+ concentrations D increases to 7.07 x 10(-9) cm2/s during the initial hours of differentiation followed by a gradual sustained decrease to values also observed in cells cultured permanently under normal Ca2+-conditions. In SCC and SVK14 cells a similar initial transient increase in lateral lipid mobility is observed upon initiation of differentiation, but, in contrast to normal keratinocytes, no sustained decrease in D is seen upon prolonged culturing under normal Ca2+ conditions. The results indicate that the deficiency of the transformed cells to respond to Ca2+-induced differentiation might involve transformation-dependent alterations in membrane structure and function.     

Immunological and structural properties of Rous sarcoma virus-transformed and tumor cells]

We compared the capacity of both normal and Rous sarcoma virus (RSV)-transformed chicken embryo fibroblast (CEF) cells as well as Rous sarcoma (RS) tumor cells to serve as targets in anti-tumor immunity assays. These studies showed that sera from tumor-bearing donors were able to stain transformed CEF more efficiently than RS cells as detected by immunofluorescence. In contrast, antiserum against the major viral glycoprotein, gp 85, stained a higher percentage of RS than transformed cells. Normal CEF cells, which served as controls, were essentially non-reactive with the immune system as judged by this type of assay. We observed that RS cells are considerably larger and contain far higher levels of protein than either normal or transformed CEF. Scanning electron microscopy revealed both the RS cells and transformed CEF to be rich in surface ruffles, blebs and microvilli as distinguished from the flattened, fusiform appearance of normal CEF cells.     

Pyruvate kinase type M2 is phosphorylated at tyrosine residues in cells transformed by Rous sarcoma virus

Chicken embryo cells (CECs) contain pyruvate kinase (PK) type M2 (M2-PK). Transformation of CECs by Rous sarcoma virus (RSV) leads to a reduction in the affinity of PK for the substrate phosphoenolpyruvate. In vitro, M2-PK can be phosphorylated at tyrosine residues by pp60v-src, the transforming protein of RSV. To study tyrosine phosphorylation of M2-PK in intact RSV-transformed cells, the protein was immunoprecipitated from 32P-labeled normal and RSV-SR-A-transformed CECs. Phosphoamino acid analysis of immunoprecipitated M2-PK revealed that M2-PK of both normal and transformed CECs contained phosphoserine and small amounts of phosphothreonine. Only M2-PK of transformed CECs contained phosphotyrosine in addition. For enzyme kinetic studies M2-PK was partially purified by chromatography upon DEAE-Sephacel and hydroxyapatite. A decreased affinity for phosphoenolpyruvate was observed 3 h after the onset of transformation using the temperature-sensitive mutant of RSV, ts-NY 68. The kinetic changes were correlated with tyrosine phosphorylation of M2-PK, but there is no direct evidence that they are caused by post-translational modification of the enzyme.     

Phenotypic changes and loss of N-CAM-mediated adhesion in transformed embryonic chicken retinal cells

Transformation of 6-d-old embryonic chicken retinal cells by Rous sarcoma virus (RSV) was found to cause significant changes in several cellular properties including adhesiveness, motility, and state of differentiation. The alterations in cell adhesivity were analyzed by means of specific antibodies to the calcium-independent neural cell adhesion molecule, N-CAM. In the RSV-transformed cells the amount of N-CAM present at the cell surface was significantly decreased relative to normal cells, as assessed by immunofluorescent staining, specific immunoprecipitation, and immunoblotting experiments. This decrease was reflected in a marked reduction in N-CAM-mediated adhesiveness measured in vitro. A different, calcium-dependent, adhesive system also present on neurons was not detectably altered by RSV transformation and, in contrast with previous studies on normal neurons, this adhesive system was detected without treatment by proteases. In culture, the transformed cells formed fewer and less compact colonies than the normal retinal cells. Observation of the RSV-transformed retinal cells by time-lapse cinematography confirmed the reduction in adhesiveness and also revealed that the transformed cells were more highly motile than their normal counterparts. In addition, RSV transformation appeared to alter the differentiation of the cultured retinal cells. Immunofluorescent staining studies indicated that in contrast to mature neurons, transformed neural retinal cells expressed the 34,000-mol-wt tyrosine kinase substrate and reduced amounts of a neuron-specific ganglioside recognized by monoclonal antibody A2B5. These characteristics are shared by untransformed glial cells. In double immunofluorescent staining experiments, many cells expressed both N-CAM and pp60src shortly after viral infection, which implies that the N-CAM-positive neuroepithelial cells were transformed by RSV. In addition, a highly purified population of N-CAM-positive neural retinal cells, selected using a fluorescence-activated cell sorter, was rapidly and extensively transformed by RSV at rates comparable to those of the unfractionated population. These results established that the transformed cells were largely derived from RSV-infected neuroepithelial cells rather than from a small population of retinal glial cells present in the primary culture. The findings suggest reconsideration of the possible origin of tumors classified by morphological criteria as derived from glia and raise the possibility that the normal homologue of pp60src may play a role in the commitment of neuroepithelial cells to neuronal or glial differentiation pathways.