Analysis of cellular expression of gangliosides by gene transfection. I: GD3 expression in myc-transfected and transformed 3Y1 correlates with anchorage-independent growth activity

Transfection of c-myc DNA into rat fibroblastic 3Y1 cell line resulted in the neosynthesis of GD3 ganglioside, as has been previously shown to occur after transfection of 3Y1 cells with adeno El (Nakakuma, H., Sanai, Y., Shiroki, K., and Nagai, Y. (1984) J. Biochem. 96, 1471-1480); in both cases, the products are expressed intranuclearly. Moreover, a clear correlation was identified between levels of GD3 expression and colony-forming activity (in soft agar) of myc-transformed 3Y1 cells, implying that GD3 plays some specific role in myc-induced transformation of 3Y1 cell line.     

The analysis of gene-regulatory mechanism of ganglioside metabolism by gene transfection

To investigate the genetic control on ganglioside metabolism, various oncogenes were directly transfected to rat 3Y1 cells, and the subsequent alteration in the ganglioside pattern was analysed. So-called "intranuclear" type oncogenes, the products of which are mainly expressed intranuclearly, ex. adeno E1 and myc, brought about specifically GD3 ganglioside with concomitant decrease in GM3. On the other hand, so-called "extranuclear" type oncogenes, the products of which are expressed extranuclearly (either on the cell membrane or in the cytoplasm), ex. ras and src, brought about SPG gangliosides but no GD3 expression was recognized. This result indicates that intracellular metabolic activities of oncogenes strongly affect the ganglioside metabolism, and that the sensitive points in the pathway of the ganglioside metabolism (synthesis) are not so various but rather restricted.     

Molecular cloning of globotriaosylceramide/CD77 synthase, a glycosyltransferase that initiates the synthesis of globo series glycosphingolipids

The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (alpha1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed alpha1, 4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids.     

A new recipient cell line for transfection of biologically active oncogenes]

The properties of the new immortalized rat cell line (REF-1) were analyzed. These cells can be used as recipient ones in transfection assays. REF-1 cells never convert spontaneously to transformed phenotype during long-term passages in vitro unlike NIH3T3 cells. This peculiarity allowed to use REF-1 cells for identification of oncogenes, which induce slow-growing tumors. The following oncogenes were used in gene transfection experiments in order to test their effects: activated human EJ-ras; N-ras; v-myc; v-mos; activated tpr-met and c-hu-met. REF-1 cells, transfected with the members of ras family; v-mos and tpr-met were found to be transformed in vitro and induce tumors in nude mice, on the contrary of c-hu-met- and v-myc-transfected cells, which are non-tumorogenic. A number of clonal cell lines carrying different oncogenes were obtained. The detailed analyses of integration and expression of exogenous sequences of different oncogenes has been presented in 18 clonal cell lines.     

Transformation of diploid human fibroblasts by transfection with the v-sis, PDGF2/c-sis, or T24 H-ras genes

Gene transfection techniques have provided powerful methods to examine the roles of cellular and retroviral oncogenes in the transformation process in rodent fibroblasts. However, the use of such techniques with diploid human fibroblasts has been limited. We have developed transfection procedures to reproducibly transfect such cells with oncogenes, and methods for the biological characterization of the transformants. We have shown that the v-sis and T24 H-ras oncogenes, as well as the platelet-derived growth factor gene (PDGF2/c-sis), are capable of inducing a transformed phenotype in normal diploid human fibroblasts, but are not capable of conferring infinite lifespan or making such cells tumorigenic.     

Altered ganglioside expression in ras-oncogene-transformed cells

Alteration of ganglioside composition in mouse BALB/3T3 cells transformed either by DNA transfection with viral K-, H-, or cellular H-ras oncogene, or by infection with the K-ras oncogene-carrying murine sarcoma virus (Ki-KSV) was studied using a highly sensitive thin-layer chromatography/enzyme immunostaining method. Marked common decreases in the content of GD3 ganglioside and the increase of its metabolic precursor GM3 were bound in BALB/3T3 cell lines transformed by either K- or H-ras oncogenes. Moreover, a common decrease or loss in the contents of "A" series ganglio-tetraose gangliosides such as GM1a and GD1a was also found in all transformed cell lines, indicating that the alteration of cellular glycosphingolipids by ras oncogenes apparently does not depend on the type of ras-concogenes (K- and H-ras).     

Immortalization by c-myc, H-ras, and Ela oncogenes induces differential cellular gene expression and growth factor responses.

Early-passage rat kidney cells were immortalized or rescued from senescence with three different oncogenes: viral promoter-driven c-myc, H-ras (Val-12), and adenovirus type 5 E1a. The normal c-myc and H-ras (Gly-12) were unable to immortalize cells under similar conditions. Quantitation of RNA in the ras-immortalized lines demonstrated that the H-ras oncogene was expressed at a level equivalent to that of the normal H-ras gene in established human or rat cell lines. Cell lines immortalized by different oncogenes were found to have distinct growth responses to individual growth factors in a short-term assay. E1a-immortalized cells were largely independent of serum growth factors, whereas c-myc-immortalized cells responded to serum better than to epidermal growth factor and insulin. H-ras-immortalized cells responded significantly to insulin alone and gave a maximal response to epidermal growth factor and insulin. Several cellular genes associated with platelet-derived growth factor stimulation, including c-myc, were expressed at high levels in the H-ras-immortalized cells, and c-myc expression was deregulated, suggesting that the H-ras oncogene has provided a "competence" function. H-ras-immortalized cells could not be morphologically transformed by secondary transfection with a long terminal repeat-c-myc oncogene, but secondary transfection of the same cells with H-ras (Val-12) produced morphologically transformed colonies that had 20- to 40-fold higher levels of H-ras oncogene expression. Thus, transformation in this system is dependent on high levels of H-ras oncogene expression rather than on the presence of activated H-ras and c-myc oncogenes in the same cell.     

Activated v-myc and v-ras oncogenes do not transform normal human lymphocytes.

Activated v-myc (pSV v-myc) and v-Ha-ras (GT10) oncogenes were introduced into normal human lymphocytes, NIH 3T3 fibroblasts, B-lymphoblastoid cells, and human epithelial cells, using a reconstituted Sendai virus envelope-mediated gene transfer technique. Efficient transfer of the plasmid in each cell type was demonstrable within 1.5 h of transfection by Southern blotting of extrachromosomal DNA extracts, which unexpectedly revealed that v-myc plasmid DNA was unstable in normal lymphocytes but not in the other cell types. The v-myc plasmid was stabilized when cotransfected into lymphocytes together with v-Ha-ras. The transfected v-Ha-ras plasmid was stable in all the cell types tested. v-myc plasmid expression was clearly detectable by 5 h in all cell types except human lymphocytes. Lymphocytes expressed v-myc when transfected together with v-Ha-ras. Transfected ras oncogene was efficiently expressed in all the cell types tested. Expression of the transfected genes increased at 24 and 48 h after transfection. Even though plasmid stability and expression were achieved in myc-ras-cotransfected lymphocytes, no effects on cellular DNA synthesis or immortalization were observed, in contrast to efficient transformation of NIH 3T3 fibroblasts by the same procedure. Our data suggest that efficient expression of transfected myc and ras oncogenes in normal quiescent human lymphocytes is not sufficient for the induction of cell growth and immortalization.     

Freeze-fracture electron microscopic studies of age-related plasma membrane changes in Sporothrix schenckii

Characteristics of the plasma membrane of Sporothrix scheckii cells as revealed by freeze-fracture techniques have been classified into eight types (Y1, Y2a, Y2b, Y3a, Y3b, Y4a, Y4b, and Y5) in yeastlike cells grown under the following two conditions: brain heart infusion agar medium at 27 degrees C, and brain heart infusion agar medium at 37 degrees C. Type Y1 cells are yeastlike cells having smooth plasma membranes without any invagination. Typical characteristics of the other types are as follows: type Y2a, smooth plasma membranes with few trenchlike invaginations; type Y2b, wavy plasma membranes with few oval or irregularly formed invaginations; type Y3a, plasma membranes with many randomly distributed trenchlike invaginations; type Y3b, plasma membranes with many cocoonlike or irregularly formed invaginations; type Y4a, plasma membranes with longer trenchlike invaginations; type Y4b, plasma membranes with irregularly formed, enlarged invaginations; and type Y5, smooth or wavy plasma membranes with aggregations of intramembranous particles and with many vacuoles between cell walls and plasma membranes or in the cytoplasm in some cells. By counting the proportion of each type of yeastlike cell under the two conditions and with different cultivation periods, it appears that plasma membrane types change as aging progresses in the following order: type Y1, Y2a, Y3a, Y4a, and Y5 in conidia and type Y1, Y2b, Y3b, Y4b, and Y5 in yeastlike vegetative cells. These observations provide us with an important advantage when studying the effects of antifungal agents on the plasma membrane of Sporothrix scheckii, as it is important to know the natural course of changes in membrane structure during aging.     

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.     

FAK mediates signal crosstalk between type II collagen and TGF-beta 1 cascades in chondrocytic cells

The purpose of this study was to evaluate the mechanism of crosstalk between the type II collagen and TGF-β1 signaling pathways in chondrocytic cells. Articular chondrocytes, isolated from porcine knee cartilage, and the SW1353 cell line were cultured on either type II collagen-coated or -uncoated plates in the presence or absence of TGF-β1. Expression of pSMAD 2, pSMAD 3, pFAK_Y397 and pFAK_Y925 in articular chondrocytes and the SW1353 cell line was analyzed by immunoblotting. Cell proliferation rates and glycosaminoglycan (GAG) content was determined after treatment with type II collagen or/and TGF-β1. For inhibition study, human FAK-specific RNA small interference (siFAK) in SW1353 cell line was performed. In this study, expression of pSMAD 2, pSMAD 3, pFAK_Y397 and pFAK_Y925 were synergistically increased by co-treatment with type II collagen and TGF-β1 in articular chondrocytes. The proliferation of porcine articular chondrocytes and GAG secretion in SW1353 cells were synergistically increased by co-stimulation with type II collagen and TGF-β1. Synergistically increased expression and nuclear translocation of pSMAD 2 and pSMAD 3 and GAG secretion of SW1353 cells were significantly inhibited by siFAK transfection. Therefore, we suggest that FAK-SMAD 2/3 mediates signal crosstalk between type II collagen and TGF-β1 and regulates GAG secretion in chondrocytic cells.     

Effects of cellular transformation on expression of plasminogen activator inhibitors 1 and 2. Evidence for independent regulation

Expression of plasminogen activators (PA) has been reported to be associated with invasive tumor growth and increased metastatic ability. In order to delineate changes in PA and PA inhibitor (PAI) expression that accompany cellular transformation, we studied oncogene-containing variants of the Rat-1 cell line. We report here that transfection of the oncogenes v-src, erbB, c-myc, v-myc, N-myc, and EJras into these cells does not result in detectable PA activity in conditioned media or cell extracts. In addition, Northern blot analysis fails to demonstrate urokinase mRNA in Rat-1 cells or transfectants. Moreover, cells transformed by EJras and v-src but not other oncogenes secrete an active placental-type PAI, PAI-2. Using inducible EJras constructs, we find that increased PAI-2 gene expression is detectable within 6-12 h after treatment with the inducing agent. Peak expression of PAI-2 mRNA is increased 10-15-fold over base line, and high levels are maintained for at least 72 h. In contrast to the results with PAI-2, secretion of endothelial-type PAI-1 into conditioned media is sharply down-regulated by several oncogenes. Thus, we have found that PAI-1 and PAI-2 are independently regulated in transformed variants of Rat-1 cells. The specific induction of PAI-2 in cells transformed by oncogenic ras and src suggests that this protease inhibitor may have a previously unsuspected role in malignancy.     

Calpain processing of brain microtubules from the Atlantic Cod,Gadus morhua

Viral infection of cultured cells with transforming viruses causes an increase in cell-surface N-linked 1-6 (GlcNAc1-6Man) branching of complex-type oligosaccharides. Similar observations have been made after transfection of cells with activated oncogenes, which is associated with an induction of tumorigenic and metastatic properties. In this study, the effects of transfection of both activated and proto-Ha-ras oncogenes into NIH3T3 cells were analyzed. The results showed that, in comparison with NIH3T3 cells, bothras transfectants have increased sensitivity to the cytotoxic action of L-PHA. An increase in 1-6 branching and an increased level of N-acetylglucosaminyltransferase V (GlcNAc-T V), the enzyme which initiates the 1-6 branching were also observed. The levels of GlcNAc-T I and 1-4 Gal-T remained unchanged in activated Ha-ras transfected NIH3T3 cells. These data suggest that a specific induction of GlcNAc-T V occurs after transfection with either the proto- or activated Ha-ras oncogenes, which is responsible for the increased 1-6 branching previously observed. (Mol Cell Biochem122: 85–92, 1993)Abbreviations Asn Asparagine - BHK Baby Hamster Kidney (cells) - 1-4 Gal-T or Gal-T 1-4 Galactosyltransferase - BSL-II Banderia Simplifolia Lectin II - CHO Chinese Hamster Ovary (cells) - ConA Concanavalin A - D-MEM Dulbecco's Modified Eagle's Medium - E-PHA Erythroagglutinin of Phytohemagglutinin - Fuc Fucose - Gn or GlcNAc N-acetylglucosamine - GlcNAc-T I N-acetylglucosaminyltransferase I - LCA Lens Culinaris Agglutinin - L-PHA Leukoagglutinin of Phytohemagglutinin - M or Man Mannose - PBS Phosphate-Buffered Saline - PL Pea Lectin - RIC Ricin - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - UDP-Gal Uridine-Diphosphate-Galactose - UDP-Gn Uridine-Diphosphate-N-acetylglucosamine - WGA Wheat Germ Agglutinin     

Synthesis of diacylglycerol de novo is responsible for permanent activation and down-regulation of protein kinase C in transformed cells

We measured the synthesis of diacylglycerol de novo in normal NIH/3T3 fibroblasts and in cells transformed by ras, src, sis and abl oncogenes. Analysis of the incorporation of glucose-derived 14C into diacylglycerol indicated that neosynthesis of diacylglycerol was constitutively active in the transformed cell lines. Elevated levels of diacylglycerol and persistent activation/down-regulation of protein kinase C reduced the binding of phorbol dibutyrate to transformed cells. This phenomenon could be reversed by blocking the glycolytic pathway, thus indicating that neosynthesized diacylglycerol was responsible for persistent activation and down-regulation of protein kinase C. In transformed cells, protein kinase C activity could not be stimulated by the addition of diolein; however, inhibition of glycolysis restored the ability of transformed cells to respond to diolein. Taken together these data indicate that constitutive synthesis of diacylglycerol de novo is responsible for activation and down-regulation of protein kinase C in transformed cells, and it may play a role in altered mitogenic signalling.     

Role of oncogenes in the regulation of MHC antigen expression.

Class I and class II major histocompatibility complex (MHC) antigens are required for CD8+ cytotoxic T cells and CD4+ helper T-cells, respectively, to recognize foreign antigen. Regulating the levels of expression of these MHC antigens regulates the T-cell responses [1]. This regulation is mainly carried out by the interferons (IFN), which are produced in the disease state. Type I IFN (IFN alpha or IFN beta; collectively 'IFN alpha beta) up-regulates class I MHC and IFN gamma up-regulates class I and class II MHC. We and others [1-3] have shown that transfection of cells with a variety of oncogenes including ras and myc affects the level of MHC antigen expression. This and other data provide evidence for a scheme in which the signal transduction mechanisms whereby IFN up-regulates MHC antigens involve several (proto) oncogenes.