Platelet-derived growth factor does not induce c-fos in NIH 3T3 cells expressing the EJ-ras oncogene.

Platelet-derived growth factor (PDGF), the calcium ionophore A23187, and the tumor promoter phorbol myristate acetate stimulated c-fos mRNA levels in control NIH 3T3 cells. However, NIH 3T3 cells transformed by EJ-ras DNA transfection, which have diminished PDGF-stimulated phospholipase C activity, showed a 95% reduction in PDGF-stimulated c-fos mRNA levels. The responses to A23187 and phorbol myristate acetate were also attenuated, but not as severely as the PDGF-mediated induction. The reduction in PDGF-stimulated c-fos induction did not appear to be a general result of cellular transformation, since src-transformed NIH 3T3 cells displayed a strong PDGF-stimulated c-fos induction. Despite the reduction in PDGF-stimulated c-fos induction, EJ-ras-transformed cells still responded mitogenically to PDGF. These data suggest that the magnitude of c-fos induction cannot be directly correlated with PDGF-stimulated mitogenesis in EJ-ras-transformed NIH 3T3 cells.     

The sensitivity to growth factors of NIH 3T3 cells transformed by the v-myc oncogene

Transformation of NIH 3T3 cells, induced by v-myc oncogene, activates a proliferative potential of the cells cultivated in the serum-free medium, and reduces the ratio of 3H-Tdr incorporation into the cells grown in the presence of 10% fetal serum in comparison to those grown in the serum-free medium. The v-myc transformed cells (NIH 3T3-v-myc) as well as the untransformed ones are very responsive to insulin. On the other hand, the epidermal growth factor, able to stimulate proliferation of NIH 3T3 cells, exert no effects on the NIH 3T3-v-myc cells. The NIH 3T3-v-myc cells cultivated in the medium, containing 2.5% human plasma enriched with thrombocytes, have the same proliferative characteristics as cells grown in the thrombocyte-free plasma. It is concluded that transformation of NIH 3T3 cells induced by v-myc oncogene may reduce a requirement for thrombocyte-released growth factors and EGF but not for insulin.     

Expression of human recombinant plasminogen activators enhances invasion and experimental metastasis of H-ras-transformed NIH 3T3 cells.

The gene transfer technique was used to examine the role of plasminogen activator (PA) in the invasive and metastatic behavior of tumorigenic cells. H-ras-transformed NIH 3T3 clonal cells producing a very low level of PA were generated and further transfected with an expression plasmid containing a cDNA sequence encoding either the urokinase-type or the tissue-type human PA. Compared with the parental transformed cells, clonal cells expressing high levels of both types of recombinant PA invaded more rapidly through a basement membrane reconstituted in vitro. Furthermore, cells expressing high levels of recombinant urokinase-type PA also caused a higher incidence of pulmonary metastatic lesions after intravenous injection into nude mice. Both activities were reduced by the serine proteinase inhibitor EACA; invasion was also suppressed by antibodies blocking the activity of human PAs and by the synthetic collagenase inhibitor SC-44463. These findings provide direct genetic evidence for a causal role of PA in invasive and metastatic activities.     

Altered expression and distribution of the cytoskeletal-associated p35 protein in NIH 3T3 cells transformed with the Harvey sarcoma virus v-ras oncogene

1. Cytoskeletal events associated with retroviral oncogene (v-ras)-mediated transformation were studied in NIH 3T3 fibroblasts and their v-ras-transfected counterparts (3T3/H-1 cells). 2. Abnormal microfilament networks seen in 3T3/H-1 cells reflected significant decreases (approximately 90%) in two cytoskeletal-associated proteins (tropomyosin-1, p35). Neither actin content nor actin mRNA levels were altered, however, v-ras transfectants. 3. p35 mRNA activity in both NIH 3T3 and 3T3/H-1 cells was similar although differential compartmentalization of p35 to the detergent-resistant cytoskeletal fraction was evident only in normal fibroblasts. 4. Proper cytoskeletal organization may be a factor in the regulation of p35 mRNA translation in situ or influence the stability of p35 independent of translational rate.     

Isolation of recessive (mediator-) revertants from NIH 3T3 cells transformed with a c-H-ras oncogene.

We have generated two serum- and anchorage-dependent revertants from NIH 3T3 cells transformed with multiple copies of the human c-H-ras oncogene. In both revertants, the c-H-ras oncogene was fully expressed. Fusion of either revertant with untransformed cells or of the two revertants with one another resulted in transformed progeny. These results indicated that the two revertants were recessive and in different complementation groups. We believe that in our two revertants some of the genes mediating the transforming activity of the c-H-ras oncogene are defective; we are attempting to identify these mediator genes.     

Hormonal regulation of expression of the oncogene v-src in mouse fibroblasts NIH 3T3

NIH 3T3 cells were transfected by plasmid containing v-src under control of hormone-regulated LTR MMTV (pMLsrc10). This plasmid caused the foci of morphologically transformed cells. The transformed cells induced rapidly growing tumours in nude mice. In the presence of dexamethasone the efficiency of NIH 3T3 cell transformation increased ten times, while tumourigenicity remained unchanged.     

Adenylate cyclase activity of NIH 3T3 cells morphologically transformed by ras genes

The observed homology between G-proteins which regulate adenylate cyclase and ras proteins and the suggested role of ras in the regulation of adenylate cyclase in yeast prompted us to examine the regulation of adenylate cyclase in three cell lines: (i) NIH 3T3 cells, (ii) NIH 3T3 cells transformed by high levels of the normal rasH gene product and (iii) NIH 3T3 cells transformed by a mutated rasH gene product. We found that the regulation of adenylate cyclase by G-proteins is identical in the three cell lines, although the response of the transformed NIH 3T3 cells to agonists is strongly attenuated. Our data suggest that mammalian ras products do not interact directly with adenylate cyclase, although their increased expression may indirectly inhibit the interaction of adenylate cyclase stimulatory receptors with G-proteins.     

Comparison of sialyl- and alpha-1,3-galactosyltransferase activity in NIH3T3 cells transformed with ras oncogene: increased beta-galactoside alpha-2,6-sialyltransferase.

Previous studies have indicated that transfection of NIH3T3 cells with the ras oncogene induced modifications of the terminal glycosylation of N-linked glycans which appeared in the early stage after transfection. These changes affected especially the terminal part of N-linked glycans which is substituted with alpha-1,3-Gal residues in NIH3T3 and with Neu5Ac residues in the ras-transformed counterpart. We have transformed NIH3T3 cells with the human c-Ha-ras oncogene, evaluated tumorigenicity and metastatic capacity in vivo and compared alpha-1,3-galactosyltransferase, alpha-2,3- and alpha-2,6-sialyltransferases activities. By using different specific acceptors, we detected the enhancement of sialic acid transfer in transformed cells while the activity of alpha-1,3-galactosyltransferase remained unchanged. We showed that the higher sialyltransferase activity was due to the increase of beta-galactoside alpha-2,6-sialyltransferase in ras-transfectant although alpha-2,3-sialyltransferase was weakly expressed in these cells. On the basis of binding of different lectins, we correlated these observations with changes of protein glycosylation. We concluded that altered glycosylation of ras-transformed NIH3T3 is the result of a competitive effect of the enzymes acting for terminal glycosylation of N-linked glycans and the reflection of the higher expression of alpha-2,6-sialyltransferase.     

Tumorigenicity of morphologically distinct transformed foci induced by 3-methylcholanthrene in BALB/c-3T3 cells

4 mm in diameter), invasiveness (smooth vs. invading margins) and other properties (piling vs. spread). In our previous report, we showed that cells from all five types grew in soft agar, transformed normal NIH 3T3 cells and formed foci on normal layer of BALB/c-3T3 cells. In this study, the neoplastic/tumorigenic potential of cells from the five different types of transformed foci was investigated in nude mice. About two million cells from each transformed focus were injected into 4-week-old nude mice. Non-transformed BALB/c-3T3 cells were used as control. The results of this study indicate that all the 45 athymic mice injected with different transformants developed tumors between 2 and 4 weeks after injection. Tumors were not observed in eight mice injected with non-transformed BALB/c-3T3 cells. All tumors were histopathologically confirmed fibrosarcomas. These findings indicate that all five morphologically different foci show tumorigenicity and that any foci of size > or =2 mm regardless of invasiveness and piling could be scored as positive during the cell transformation assay.     

Transformation of NIH 3T3 cells by enhanced PAR expression

Prostate androgen regulated (PAR) is a 1038bp novel gene located on chromosome 1 in epidermal differentiation complex. The gene is ubiquitously expressed in normal tissues and is overexpressed in most of their malignant counterparts. PAR cellular function is unknown. Here we report the effect of increased PAR expression induced by transfection of PAR cDNA on NIH3T3 cell phenotype. PAR-NIH3T3 transfectants expressing 3- to 4-fold higher PAR levels compared to controls grew faster in tissue cultures, formed colonies in soft agar, and exhibited a shortening of G1 and S phases of cell cycle and formed tumors in SCID mice. Transfection of NIH3T3 cells with increased ectopic PAR expression with a 22 mer oligonucleotide in antisense orientation with PAR mRNA abrogated their ability to form colonies in soft agar. The data presented here along with our previously reported results on DU145 cells transfected with antisense PAR cDNA suggest that PAR gene behaves like a proto-oncogene.     

GTP-binding proteins and adenylate cyclase activity in v-Ki-ras transformed NIH/3T3 fibroblast cells

To identify the role of ras oncogene and p21 in the coupling mechanism of GTP-binding proteins to adenylate cyclase, we used v-Ki-ras transformed NIH/3T3 fibroblast cells. In the previous study, we investigated that NaF, cholera toxin and forskolin remarkably enhanced the adenylate cyclase activity in transformed cells compared to normal NIH/3T3 cells. In the present study, adenylate cyclase was more enhanced by GTP gamma S in transformed cells than in normal cells. It was considered that p21 plays enhancing role in coupling of GTP-binding proteins to adenylate cyclase. Further, as measured by the degree of [32P] ADP-ribosylation of GTP-binding proteins by cholera toxin and pertussis toxin respectively, the amount of Gs (46 kDa) was almost equal in both cells, while the amount of Gi (41 kDa) in transformant was about one third of that in normal cells. This difference seems to be reflected in either the biological situations or the quantities of Gi. Our data suggest that v-Ki-ras transformation resulted in the decrease of Gi protein so that the inhibitory regulation on adenylate cyclase relatively becomes low and then stimulatory influence of Gs seems to be enhanced.     

Molecular cloning of the 25 kbp region upstream of exon 0 of the human Ki-ras oncogene and its conservation in transformed mouse NIH 3T3 cells

Human sequences associated with the Ki-ras oncogene of the mammary tumour cell line, H-466B have been cloned from a tertiary NIH3T3 mouse transfectant. These sequences are located 5' upstream of exon 0 of the Ki-ras oncogene, span over 25 kbp of DNA and are conserved in half of the primary transfectants obtained with the Ki-ras gene of different types of tumours. No gross alterations were observed in the sequences upstream of the Ki-ras gene. The partial or total deletion of these sequences in the other half of primary transformants argues that they are not absolutely required for the transforming activity of the Ki-ras oncogene. The even distribution of the human-mouse junction points in primary transformed mouse cells suggests the absence of a specific region of recombination in the 5' flanking region of Ki-ras.     

Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ inter- acted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.     

Potentiation of DNA mediated gene transfer in NIH3T3 cells by activators of protein kinase C

The mechanisms involved in the translocation of exogenously added genetic information through the cellular cytoplasm and into the nucleus are essentially unknown. Several trans-cytoplasmic translocation systems operate within cells to transport information received by the plasma membrane into the nucleus. Protein kinase C may be functionally involved in many of these translocation mechanisms. In order to explore the involvement of protein kinase C activation in the cytoplasmic translocation of DNA, NIH3T3 fibroblasts were transfected using the calcium-phosphate co-precipitation method with a plasmid containing the lacZ gene and treated with tetradecanoylphorbol 12,13-acetate (TPA) or 1,2-dioctanoylglycerol (DiC8). Addition of TPA or DiC8 immediately after glycerol shock resulted in a 5-7-fold increase in the number of cells expressing beta-galactosidase as well as a concomitant increase in the total amount of beta-galactosidase activity in the population during periods of transient and stable expression. TPA added at later times resulted in lesser increases in the efficiency of transfection. In contrast, TPA added at the time of addition of the calcium-phosphate precipitate inhibited transfection. In support of a role for protein kinase C activation in enhancing DNA transfection, the TPA analog 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, was ineffective at enhancing transfection. Furthermore, treatment of cells with the protein kinase C inhibitor sphingosine blocked the TPA-mediated increase in transient and stable expression. The results suggest that protein kinase C activation enhances transfection of exogenous DNA through an as yet unknown mechanism.     

Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells

The epidermal growth factor receptor (EGFR) gene is frequently amplified and/or overexpressed in human malignancies. To investigate the biological effects of its overexpression, we constructed a eukaryotic vector containing human EGFR cDNA. Introduction of this construct led to reconstitution of functional EGF receptors in NR6 mutant cells, which are normally devoid of this receptor. Transfection of NIH 3T3 resulted in no significant alterations in growth properties. However, EGF addition led to the formation of densely growing transformed foci in liquid culture and colonies in semisolid medium. NIH 3T3-EGFR clonal lines, which expressed the EGF at 500- to 1000-fold levels over control NIH 3T3 cells, demonstrated a marked increase in DNA synthesis in response to EGF. Thus EGF receptor overexpression appears to amplify normal EGF signal transduction. Finally, high levels of EGFR expression, which conferred a transformed phenotype to NIH 3T3 cells in the presence of ligand, were demonstrated in representative human tumor cell lines that contained amplified copies of the EGFR gene.     

The enhanced transfer of drug-resistant genes in NIH-3T3 cells transformed by the EJras oncogene

The spontaneous transfer of drug resistance genes has been shown to take place between cultured mammalian NIH-3T3 cells and occurs with a hierarchy of transfer efficiencies, transformed cells being more efficient than non-transformed cells. This experiment was accomplished by co-cultivating two NIH-3T3 sublines, each transfected by standard plasmid methods with a different drug resistance gene, subjecting the mixed population to double selection by adding both drugs to the mixed cell culture, and isolating single cells which were resistant to both drugs. The genes used were the neo gene and gpt gene which conferred resistance to the drugs G418 and mycophenolic acid, respectively. DNA analysis confirmed the presence of both resistance genes in the cells which were resistant to both drugs. The mechanism of this gene transfer was by cell fusion rather than by chromosomal DNA uptake. The efficiency of gene transfer, as indicated by the number of double-resistant colonies standardized by number of cells cultured, was much higher between two sublines of cells transformed by the EJras oncogene than between one transformed and one non-transformed subline, which in turn was higher than between two non-transformed sublines. The higher efficiency of gene transfer between the transformed cells also occurred when these cells were injected into nude mice, thus demonstrating that the same process occurred in vivo. It would appear that drug resistance genes may be transferred spontaneously in cultured mammalian cells by cell fusion, and that transformed cells have a higher efficiency of gene transfer compared to non-transformed cells.