Nontransformed Phenotype Research Articles

Membrane changes associated with the temperature-sensitive P85 gag-mos-dependent transformation of rat kidney cells as determined by dielectrophoresis and electrorotation

Conventional dielectrophoresis (cDEP) and electrorotation (ROT) measurements have been used to determine the dielectric properties of a clone of normal rat kidney cells, designated 6m2, that exhibits a transformed phenotype at 33°C and a non-transformed phenotype at 39°C. cDEP measurements of the crossover frequencies at which individual 6m2 cells experienced zero cDEP force performed as a function of the conductivity of the suspension medium revealed that, in response to a temperature shift from 33°C to 39°C for 24 h, the mean specific cell membrane capacitance and conductance fell significantly ( P < 0.01) from 42.3 (±1.3) to 30.3 (±2.9) mF/m 2 and 743 (±422) to 567 (±326) S/m 2, respectively. ROT analyses demonstrated a similar reduction for the membrane capacitance from 37.2 (±7.3) to 27.4(±6.1) mF/m 2, and also showed that accompanying changes in the mean internal electrical conductivity and dielectric permittivity of the cells were insignificant. Scanning electron microscopy was used to examine the surface morphology of the cells and, in agreement with our previous reports for leukemia cells, the observed membrane capacitance values correlated closely with the morphological complexity of the cell membrane surface. The observed changes in the membrane dielectric properties are discussed in terms of their biological significance and their relationship to previously-detected changes in cell surface charge.

Drug-induced reversion of progression phenotype is accompanied by reversion of AP-1 phenotype in JB6 cells.

Transformed JB6 cells can be stably reverted to nontransformed phenotype by AP-1 inhibiting gluccorticoid fluocinolone (FA) and cAMP elevator forskolin (FN), yielding stable revertants of promotion resistant (P-) and promotion sensitive (P+) phenotypes. AP-1 activity of nontransformed P- and P+ revertant clones was decreased under a variety of experimental conditions compared with their transformed counterparts. Moreover, AP-1 activity in P+ cells under anchorage-independent conditions was induced by 12-0-tetradecanoyl-phorbol-13-acetate (TPA) while AP-1 activity in the reverted P- cells was not induced, just as observed for the original P+ and P- variants. Taken together these data suggest that changes in AP-1 activity may be one key mediator not only of forward progression but also of reversion of tumor cells to nontransformed phenotype. In addition, the higher transfection efficiency of the new reverted P- and P+ cells renders them useful for studying the role of transcription factors in tumor promotion.

Passage of X-ray-induced immortal, non-transformed phenotype by DNA-mediated transfection

To better understand the molecular basis of X-ray-induced carcinogenesis, the immortalization step of this multistep process was examined. Primary rat embryo cells were X-irradiated in vitro and six clones were isolated. Three of these, one designated X-REF-23, were immortal and non-transformed. Transfection of high molecular weight DNA from rat X-REF-23 cells into primary mouse cells yielded two immortal and non-transformed mouse clones, 1K and 2I. Using a 2.3 kb rat-specific repetitive sequence as probe, 1K and 2I were demonstrated to contain rat DNA. This transfected DNA was not any of the known immortalization-associated proto-oncogenes. DNA from 1K was then transfected into primary mouse cells, with or without co-transfection of pSV2 neo DNA. Six immortal mouse clones were isolated and confirmed to contain rat sequences. In conclusion, the immortal phenotype can be transferred by DNA transfection.

Platelet-derived growth factor (PDGF) B and A homodimers transform murine fibroblasts depending on the genetic background of the cell.

The endogenously overexpressed c-sis (platelet-derived growth factor (PDGF) B-chain) proto-oncogene and its viral counterpart v-sis effectively transform NIH 3T3 cells by an autocrine mechanism, whereas the PDGF A-chain gene does not. However, PDGF B continuously cultured with confluent NIH 3T3 cells fails to induce phenotypic transformation as would be predicted by the autocrine model. We now have established conditions in which subconfluent, logarithmically growing NIH 3T3 cells are efficiently transformed by exogenous PDGF B but not PDGF A. Clonally selected cells from transformed foci remain transformed when continuously cultured with PDGF B but revert to the non-transformed phenotype without PDGF B or if PDGF A is substituted for PDGF B, suggesting that PDGF B complements a genetically stable NIH 3T3 cell phenotype to induce transformation and initiates a signal different from that of PDGF A required to induce transformation. Neither PDGF A nor PDGF B transform normal rat kidney (NRK) cells under identical conditions of culture. However, we now demonstrate that exogenous PDGF A and PDGF B independently transform NRK cells that endogenously overexpress the anti-apoptotic bcl-2 gene. These results suggest that both PDGF A and PDGF B effectively transform murine fibroblasts with a compatible genotype, that transformation by the PDGF isoforms requires complementation with a compatible genotype in a multistep transformational process, and that the autocrine mechanism is required but not sufficient for transformation of murine fibroblasts by PDGF.

Regulation of gamma‐glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells

Rat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII-related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen-coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three-dimensional structures. The activity of the blood-brain barrier-associated enzyme, gamma-glutamyl transpeptidase (gamma GTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel-related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three-dimensional structures, but every cell inside the aggregates still expressed gamma GTP and ALP activities. Such structures, associated with high levels of gamma GTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood-brain barrier-related gamma GTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood-brain barrier.

Mechanisms of reversion.

Reversion of transformed cells provides biological system useful in multiple areas of cancer research. First, reversion can be induced by inhibiting the activities of specific oncogenes or replacing intact tumor suppressor genes, and such experiments yield important insights into the roles of these genes in carcinogenesis and normal growth regulation as well as the functional relationships between these genes and other known genes. Second, novel genes involved in growth regulation can be discovered by isolating revertants from transformed cells after mutagenesis or DNA transfection and then characterizing the genes responsible for the non-transformed phenotype. Third, potential anti-cancer drugs can be screened by reversion assay using cells transformed by specific oncogenes. The novel genes and chemicals discovered through these studies serve as valuable tools that help our understanding of how cells regulate their growth and differentiation.

Transformation of NIH/3T3 to Anchorage Independence by H- Ras Is Accompanied by Loss of Suppressor Activity

Despite their familiar sensitivity to transformation by dominant-acting ras oncogenes, NIH/3T3 cells carry a ras suppressor. When tested by cell fusion they were able to suppress the anchorage-independent phenotype of both mouse and human cells transformed by activated H- ras or N- ras. This suppression occurred without a decrease in expression of the activated ras oncogene. Ras-transformed NIH/3T3 clones cured of their oncogene by benzamide treatment reverted to a non-transformed phenotype, but had lost the ability to suppress other ras transformants, indicating that their initial transformation was accompanied by suppressor loss. In hamster cells an active ras oncogene increased the rate of chromosome segregation by >100-fold. These results suggest that in vitro transformation of NIH/3T3 cells by ras may be more similar to multistep in vivo tumor development than previously suspected, involving not only expression of an active oncogene but also loss of a suppressor activity, perhaps induced by the clastogenic oncogene.

Response of FR3T3 cells transformed by HA‐ras oncogene and epidermal growth factor gene to differentiation induction by N,N‐dimethylformamide

Treatment of chemically transformed fibroblasts with N,N-dimethylformamide (DMF) results in the restoration of a nontransformed phenotype. In an attempt to identify more precisely the mechanisms by which DMF reverses the transformed phenotype, the effects of DMF on fibroblasts which were transformed by a single gene--specifically a synthetic epidermal growth factor (EGF) gene or the Ha-ras oncogene--were examined. The constitutive expression of either the Ha-ras oncogene or the EGF gene in FR3T3 fibroblasts resulted in cellular transformation. The effect of the differentiation-inducing agent DMF on several properties of these transformed cell lines was examined. The EGF-transfected cells were much more responsive to DMF than the ras-transfected cells. DMF treatment of the EGF-transfected cells resulted in the inhibition of anchorage-independent growth, the restoration of a normal cellular morphology and growth rate in monolayer culture, and the down-regulation of the proliferation-associated nucleolar protein B23. DMF treatment had a much slighter effect on growth of the ras-transfected cells in monolayer culture or under anchorage-independent growth conditions. The high proliferation rate of the ras-3 cells was associated with elevated expression of protein B23. The 19-3 cells, but not the ras-3 cells, expressed cell-surface fibronectin. Treatment of the ras-3 cells with DMF did not restore fibronectin expression. The binding of EGF was increased 3-fold in the EGF-transfected cells and decreased 20-fold in ras-transfected cells, but in neither case did DMF alter EGF binding. DMF treatment increased the secretion of EGF in the 2 transfected lines as well as in control cells. These results suggest that the aberrant-growth control in the EGF-transfected cells, but not in the ras-transfected cells, could be modulated by DMF and that the aberrant-growth control mechanisms were different in these 2 cell types.

Re-transformation of non-transformed hybrids between c-myc-activating mouse plasmacytoma cells and normal fibroblasts by transfection with activated c-Ha-ras but not c-myc

In a mouse plasmacytoma S194, c-myc oncogene is rearranged with Ig gene by chromosomal translocation and is consequently activated. We previously reported that transformation of phenotype and expression of rearranged c-myc were repressed in independently isolated hybrid clones, I-1 and IV-10, between S194 and normal fibroblasts. In order to investigate the relationship between transformation of phenotype and oncogene expression, transcriptionally enhanced c-myc or activated c-Ha-ras was transfected into I-1 or IV-10I, a subclone of IV-10. Transfectants expressing high levels of c-myc were found to retain the non-transformed phenotypes. On the other hand, transfectants expressing activated c-Ha-ras showed the transformed phenotypes. These results suggest that enhanced expression of c-myc is not sufficient for re-transformation of the non-transformed hybrid clones between c-myc-activating plasmacytoma cells and normal fibroblasts, but expression of activated c-Ha-ras could diminish or overcome the tumor-suppressive activity of normal fibroblasts.

EGF-Receptor and Extracellular Matrix Changes in Mouse Pulmonary Carcinogenesis

Malignant Balb/c mouse lung cell clones related to alveologenic carcinoma exhibited low levels of epidermal growth factor (EGF) receptor activity compared to nonmalignant cell clones. Immunoprecipitation of cell homogenates and immunohistochemistry on urethane-induced lung tumors suggest that the absence of activity reflects decreased amounts of EGF receptor protein. Low levels of EGF receptor alone cannot cause neoplastic transformation, since a nonneoplastic cell cone, B5D3, exhibited low levels of EGF receptor despite its nontransformed phenotype. The reduced levels of EGF receptor in malignant clones have been mimicked by long-term (12 h) treatment of a nontransformed cell clone with 200 nM phorbol dibutyrate. The detection of mutated ras oncogene in the transformed cell lines, taken together with the EGF receptor findings, suggests that more than one alteration in the signal transduction pathway may be necessary for transformation in alveologenic adenoma and carcinoma cell systems. A further phenotypic feature of transformation, reduced expression of the extracellular matrix proteins fibronectin and laminin, may be mediated at the transcriptional level.

Retrovirus-Mediated Transfer of an Adenovirus Gene Encoding an Integral Membrane Protein Is Sufficient To Down Regulate the Receptor for Epidermal Growth Factor

We have used retrovirus-mediated gene transfer to introduce sequences encoding a 10,400-molecular-weight (10.4K) adenovirus protein previously shown to down regulate the receptor for epidermal growth factor (EGF) into two murine cell lines that possess human EGF receptors (EGF-Rs). Assays for receptor expression showed that acute infection resulted in rapid, constitutive down regulation of the EGF-R via a pathway that appears to be endosome mediated. This represents the first demonstration that 10.4K expression in the absence of other virus-encoded proteins is sufficient to elicit this response. The usefulness of this approach for the study of 10.4K-mediated signal transduction in cells with a nontransformed phenotype is discussed.

Retrovirus-mediated transfer of an adenovirus gene encoding an integral membrane protein is sufficient to down regulate the receptor for epidermal growth factor.

We have used retrovirus-mediated gene transfer to introduce sequences encoding a 10,400-molecular-weight (10.4K) adenovirus protein previously shown to down regulate the receptor for epidermal growth factor (EGF) into two murine cell lines that possess human EGF receptors (EGF-Rs). Assays for receptor expression showed that acute infection resulted in rapid, constitutive down regulation of the EGF-R via a pathway that appears to be endosome mediated. This represents the first demonstration that 10.4K expression in the absence of other virus-encoded proteins is sufficient to elicit this response. The usefulness of this approach for the study of 10.4K-mediated signal transduction in cells with a nontransformed phenotype is discussed.

Glucocorticoid dexamethasone reversibly complements EJ-RAS oncogene to transform mouse embryo BALB-3T3 cells.

EJ-A is a Balb-3T3 transfectant cell line that bears a small number of EJ-ras oncogene copies/cell, has low EJ-ras expression, and resembles the parental cell line in displaying a non-transformed phenotype. The glucocorticoid hormone dexamethasone reversibly induces transformation traits in EJ-A cells, namely: 1) morphological transformation; 2) increased growth rate and saturation density; 3) reduced G1 length; and 4) independence of the FGF requirement to initiate DNA synthesis. Western blot analysis revealed that dexamethasone does not increase the p21ras protein intracellular level. beta-IFN, added to the culture medium, does not suppress the dexamethasone-induced growth stimulation and morphological transformation. Therefore, glucocorticoid hormones can complement low EJ-ras expression to transform Balb-3T3 cells, by a mechanism that is likely to be independent of p21ras increase and beta-IFN decrease.

Over expression of proto-oncogenes: ki-ras, fos and myc in rat liver cells treated in vitro by two liver tumor promoters: phenobarbital and biliverdin

Among the liver cell strains established in the laboratory from the liver of 10-day-old rats, some of them (FV) underwent spontaneous neoplastic transformation after a number of subcultures. However one (Cl 3) had maintained a non-transformed phenotype (persistence of contact inhibition, lack of growth in soft agar and of gamma glutamyltranspeptidase activity). These cells were grown either for a short time (48 h) or a longer time (3 weeks) in the presence of two liver tumor promoters: phenobarbital (0.2 × 10 −3 M) or biliverdin (10 −6 M). Total RNA was analysed by dot blot and Northern blot, then hybridized with ki-ras, fos and myc probes, previously labelled with 32P by nick translation. The three oncogenes were well expressed by the two strains but while an over-expression was observed for the Cl 3 non-transformed cells when they were grown in presence of phenobarbital or biliverdin, the FV-transformed cells were not sensitive to the two promoters.

Transforming growth factor-β inhibition of epithelial cell proliferation linked to the expression of a 53-kDa membrane receptor

Abstract Cells whose proliferation is blocked by transforming growth factor-beta (TGF-beta) express three distinct surface glycoproteins of 53, 73, and 300 kDa that bind TGF-beta with high affinity, but whose function is unknown. We have isolated two classes of chemically-induced Mv1Lu epithelial cell mutants resistant to growth inhibition by TGF-beta. Class R mutants have selectively lost expression of the 53-kDa (type I) TGF-beta-binding protein. They have also lost the ability to respond to TGF-beta with elevated fibronectin expression and cell flattening. Class S mutants bind normally but do not respond to TGF-beta. TGF-beta-resistant mutants retain a contact inhibited, nontransformed phenotype. The properties of S mutants suggest that they are defective in the TGF-beta signal transduction mechanism, while the results with R mutants identify the type I TGF-beta-binding protein as the receptor involved in mediating TGF-beta actions on cell adhesion and proliferation.

Superoxide dismutase activities of differentiating clones from an immortal cell line.

We have studied superoxide dismutase (SOD) levels in the X-REF-23 rat embryo fibroblast cell line. X-REF-23 is an immortal cell line that maintains a nontransformed phenotype throughout its known lifespan. Low-passage X-REF-23 cells undergo spontaneous differentiation into muscle and adipose cells, while high-passage X-REF-23 cells undergo little or no differentiation. SOD activities were measured in subclones of X-REF-23, which differentiate into muscle (AMC subclone) or adipose (AMB-J) cells, as well as the parental nondifferentiating X-REF-23 cells. Total SOD activity increased in all three cell lines with time in culture. Cu-ZnSOD was induced in the AMB-J and the X-REF-23 cells with time in culture, whereas the AMC cells showed no induction. MnSOD activity was induced during the time period in which differentiation occurred in the two differentiating clones. In contrast, MnSOD was not induced in this time period in the nondifferentiating X-REF-23 cell line. However, MnSOD activity was induced in the latter cell line at a much later time. Levels of immunoreactive MnSOD correlated quite well with MnSOD activity in all three cell lines. The nondifferentiating X-REF-23 cells, but not the two differentiating cells lines, showed a large increase in cell organelles with time in culture. In particular, an increase in very small mitochondria was observed; these mitochondria often showed evidence of disorganization.

C‐myc expression and transformed phenotypes in hybrid clones between mouse plasmacytoma S194 cells and normal spleen cells or fibroblasts

Expression of the rearranged c-myc oncogene and transformed phenotypes was investigated in 2 different types of somatic cell hybrid clones between a BALB/c mouse plasmacytoma line (S194) and normal allogeneic spleen cells or fibroblasts. In the parental S194 cells, one allele of the c-myc was rearranged and its 5'-flanking region was partially deleted by recombination with the immunoglobulin C alpha gene. Due to this recombination, S194 cells expressed approximately 20-fold higher than normal spleen or fibroblast levels of c-myc transcripts from the rearranged allele, which are smaller than normal germ-line 2.4-kb c-myc transcripts, but they expressed the same low levels of 2.4-kb c-myc transcripts from the non-rearranged allele as compared with normal spleen cells or fibroblasts. All the hybrid clones retained both the rearranged and the non-rearranged c-myc. The hybrid clones between S194 and normal spleen cells showed transformed phenotypes and expressed the same high levels of rearranged c-myc transcripts and low levels of the non-rearranged c-myc transcripts as the parental S194 cells. On the other hand, the hybrid clones between S194 cells and normal fibroblasts showing non-transformed phenotypes inhibited expression of the rear-ranged c-myc to undetectable levels but expressed the non-rearranged c-myc transcripts at low levels. A hybrid clone between S194 cells and normal fibroblasts showing transformed phenotypes also exhibited the same pattern of c-myc expression as the non-transformed hybrid clones. These results indicate that expression of the rearranged c-myc in S194 mouse plasmacytoma cells is modulated in different ways in different components of cell lineages, although the correlation between the levels of rearranged c-myc transcripts and the transformed phenotypes in the hybrid clones was not absolute.

Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease.

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

Use of a Cloned Multidrug Resistance Gene for Coamplification and Overproduction of Major Excreted Protein, a Transformation-Regulated Secreted Acid Protease

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

Cloning and expression of the gene for the major excreted protein of transformed mouse fibroblasts. A secreted lysosomal protease regulated by transformation.

The major excreted protein (MEP) of mouse fibroblast cells is the 39,000 Mr precursor to a lysosomal acid protease (cathepsin L) induced by malignant transformation, growth factors, and tumor promoters. We have cloned and characterized the gene for MEP from NIH-3T3 cells. This cosmid clone (pcosMMEP), containing the unique 12,000-base pair mouse MEP gene, has been transfected into monkey kidney (CV-1) cells and human epidermoid carcinoma (A431) cells. The stable A4MEP transfectants produce mouse MEP that is an active cathepsin which is secreted, glycosylated, and processed intracellularly to lower molecular weight forms as in the wild-type NIH-3T3 cells. The CVMEP cells (nontransformed phenotype) produce quantities of mouse MEP similar to that found in NIH-3T3 cells, whereas the A4MEP cells (transformed phenotype) produce greater amounts of MEP similar to the levels seen in Kirsten virus-transformed NIH-3T3 cells. The MEP mRNAs from both mouse cells and stably transfected human cells are the same size and have the same single major site for initiation of transcription, indicating that the cloned mouse MEP promoter is active in transfected cells.