Normal Rat Embryo Fibroblasts Research Articles

Cytotoxicity of purified methionine γ- lyase produced by Pseudomonas putida on several cell lines

Many human malignant cell lines and primary tumours have absolute requirements for L-methionine (1). Under normal circumstances methionine comes from dietary proteins, most normal tissues can also synthesize methionine from either homocysteine or methylthioadenosine (2). Upon L-methionine depletion, L-methionine-dependent cancer cells are not able to divide and became arrested in the late S/ G2 phase of the cell cycle (3).Methionine cleaving enzyme (methionine γ- lyase) has been found to be an effective antitumour agent in vitro as well as in vivo (4). Methionine γ- lyase is a pyridoxal 5-phosphate dependent enzyme that catalyzes the α, γ- elimination of L-methionine to α-ketobutyrate, methanethiol and ammonia (5). Rhabdomyosarcoma (RD) cell line at passage 65, AhmedMohammed-Nahi-2003(AMN3) cell line at passage 180, AMGM (Ahmed – Majeed-Glioblastoma-Multiform) cell line at passage 65 and normal Rat Embryo Fibroblast (REF) cell line at passage 56, kindly provided by Iraqi center for cancer and medical genetics research (ICCMGR) were cultured in PRMI 1640 medium supplement with 10% heat inactivated fetal calf serum and incubated at 37 ºC with 5% CO2. Examination of cytotoxicity of methionine γ- lyase Cytotoxicity was determined with the crystal violet stain as previously described (7). In briefly , 1x105 cell/ml was seeded onto 96-well culture plates in 200 µl RPMI 1640 medium and incubated until the cell reached confluent monolayer(vary according to the kind of cell-line). After incubation period the medium was removed and 200 μl of various concentrations (1000, 500,250 and 125 μg/ml) from methionine γ- lyase were added to the plate by using 3- well replicates for each concentration. The plates were incubated at 37ºC for 96 hr. The control (cancer cell lines without treatment with enzyme) (3-well replicates) was treated with 200 μl of serum free medium and incubated at 37ºC for 96 hr. At the end of incubation period, the enzyme and medium was removed from plate and washed with PBS to remove unattached (dead) cells. Two hundred μl of crystal violet was added to each well and left for 20 min at 37ºC.The stain was removed by washing with tap water several times, and the plates were left to dry. After drying, each plate was read by using ELISA microplate spectrophotometer at 492nm wave length. The percentages of Inhibitory Rate (IR %) were estimated (8).

Role of the TCF phosphorylation state and the chromatin structure in the negative transcription regulation of the c-fos proto-oncogene in E1A + c-Ha-ras transformed cells

As compared with normal rat embryo fibroblasts (REF), c-fos transcription is suppressed in REF transformed with the E1A and cHa-ras complementing oncogenes. Negative regulation of the fos promoter is due to the serum-responsive element (SRE) constantly bound with the serum response factor (SRF) and with the ternary complex factor (TCF), which form the SRF/SRF/TCF complex. Possible mechanisms of the c-fos suppression were studied, including changes in the TCF content and phosphorylation and replacement of TCF by repressor proteins. In addition, the transcription factors were tested for DNA-binding activity, and c-fos transcription was analyzed by RT-PCR in various cell culture conditions and in the presence of phosphatase and histone deacetylase inhibitors. The c-fos suppression in transformed REF was explained by formation of a transcriptionally inactive chromatin structure in the fos promoter region as a result of histone deacetylation, rather than by insufficient TCF phosphorylation.

Antisense inhibition of the PTI-1 oncogene reverses cancer phenotypes.

The genetic alterations and molecular events mediating human prostate cancer development and progression remain to be defined. Rapid expression cloning and differential RNA display detect a putative oncogene, prostate tumor-inducing gene 1 (PTI-1), that is differentially expressed in human prostate (as well as breast, colon, and small cell lung) cancer cell lines, patient-derived prostate carcinomas, and blood from patients with metastatic prostate cancer. PTI-1 consists of a unique 5' untranslated region (5' UTR) with significant sequence homology to Mycoplasma hyopneumoniae 23S ribosomal RNA juxtaposed to a sequence that encodes a truncated and mutated human elongation factor 1alpha (Trun-EF). Stable expression of a nearly full-length 1.9-kb PTI-1 gene, but not the separate PTI-1 5' UTR or Trun-EF region, in normal rat embryo fibroblast cells, CREF-Trans 6, induces an aggressive tumorigenic phenotype in athymic nude mice. Blocking PTI-1 expression with antisense PTI-1 results in reversion of transformed PTI-1-expressing cells to a more normal cellular morphology with suppression in both anchorage-independent growth and tumorigenic potential in athymic nude mice. These findings document that PTI-1 is indeed an oncogene, and directly blocking PTI-1 expression can nullify cancer phenotypes. In these contexts, PTI-1 not only represents a gene with discriminating diagnostic properties but also may serve as a target for the gene-based therapy of human prostate and other cancers.

Induction of matrix metalloproteinase 9 expression in breast carcinoma cells by a soluble factor from fibroblasts.

Tumor-stromal interactions appear to play an important role in the induction of metalloproteinase expression in malignant tumors. We describe a tissue culture system in which expression of MMP-9 (gelatinase B or the 92 kDa type IV collagenase/gelatinase) was induced by co-cultivation of fibroblasts with breast cancer cell lines. While neither the breast cancer cells nor the normal rat embryo fibroblasts made MMP-9 alone in culture, human MMP-9 was made in the co-cultures. The MMP-9 was secreted in a latent form. The induction occurred at least in part through increases in the MMP-9 mRNA levels in the breast cancer cells. These increases did not appear to require protein synthesis. Conditioned medium from the fibroblasts could duplicate the induction of MMP-9 in the breast cancer cell lines. The active factor in the medium was inactivated by heat or by trypsin suggesting that it was a protein. This protein was in the size range of 30-100 kDa. Thus, fibroblasts could secrete a factor which was able to regulate the expression of MMP-9 in breast cancer cells.

Neoplastic Transformation of Normal Rat Embryo Fibroblasts by a Mutated p53 and an Activated ras Oncogene Induces Parathyroid Hormone-related Peptide Gene Expression and Causes Hypercalcemia in Nude Mice

Parathyroid hormone-related peptide (PTHRP) is a 141-amino acid protein identified in various carcinomas associated with humoral hypercalcemia of malignancy (HHM). Although the causal role of PTHRP in HHM syndrome has been established, the molecular and cellular mechanism by which PTHRP gene is overexpressed in certain malignancies remains unknown. We have demonstrated in the present study that PTHRP secretion was markedly induced concomitantly with the formation of transformed foci after normal rat embryo fibroblasts (REFs) were co-transfected with an activated ras (ras) and a mutated form of p53 (p53-mt) genes. In either ras- or p53-mt-transfected (nontransformed) cells, only modest or barely detectable secretion of PTHRP was observed, respectively. Northern blot analysis revealed that PTHRP mRNA was markedly induced in fully transformed cells 11 days after transfection with both ras and p53-mt genes. Inhibition of RNA synthesis with actinomycin D resulted in almost complete disappearance of PTHRP mRNA at 2-3 h, suggesting a transcriptional mechanism. Transient transfection experiments revealed that PTHRP promoter activity was induced in ras + p53-mt transfectants. REFs transformed by ras and p53-mt genes and thereby induced to secrete PTHRP in vitro produced aggressively growing tumors associated with HHM syndrome when injected into nude mice. These results suggest that activation of PTHRP gene is closely related to malignant transformation of normal mammalian cells and that ras and p53 may be important regulators of PTHRP gene transcription. The transfection-focus formation system of REFs should provide an excellent model to study the molecular and cellular mechanism underlying concomitant overexpression of PTHRP gene with carcinogenesis.

Altered ganglioside composition in virally transformed rat embryo fibroblasts

The composition of gangliosides was examined in a normal rat embryo fibroblast cell line (REF52) and in two viral transformants: a polyoma transformant (REF52-PyMLV) and a simian viral 40 transformant (REF52-SV40). The distribution of gangliosides in the cell lines was determined using gas-liquid chromatography and high-performance thin-layer chromatography. N-acetylneuraminic acid was the predominant sialic acid species detected in the three cell lines. The total ganglioside concentration (μg/100 mg dry weight of cells) in the normal, PyMLV, and SV40 lines was 144.7 ± 10.4, 153.8 ± 9.2, and 86.1 ± 6.8, respectively. Gangliosides GM3, GM2, GM1, and GD1a were the major species in the normal and transformed lines. The distribution of these gangliosides, however, differed markedly between the normal and the transformed lines and also between the transformed lines themselves. The transformed cells also differed from the normal cells in growth rate, morphology, and social behavior. The cell line with highest GM3 content (PyMLV) formed islands, whereas the normal and SV40 cell lines, which had lower GM3 levels, grew as monolayers. The findings suggest that PyMLV and SV40 transformation can have multiple and different effects on cellular ganglioside distribution and growth behavior.

Molecular and functional analysis of tumor-suppressor genes by transfection.

The transformed and tumorigenic phenotype of H-ras transfected rat FE-8 cells can be suppressed by cell fusion with normal rat embryo fibroblasts. Transfection into FE-8 cells of DNA prepared from normal human placenta followed by selective elimination of tumorigenic transfected cell clones resulted in the isolation of phenotypically normal revertants. These cells exhibited a fibroblastlike, normal morphology; were anchorage-dependent; and were unable to proliferate in medium with reduced serum concentrations. Their tumorigenicity was also reduced. The suppressed phenotype has been transferred in a second transfection cycle. Human repetitive DNA sequences were detected in secondary transfectant DNA. A putative human suppressor gene, designated NTS-1, has been molecularly cloned. Reintroduction of cloned NTS-1 sequences into FE-8 cells resulted in suppression of the neoplastic phenotype in spite of a high ras expression.

Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts.

We have used immunocytofluorescence techniques to determine the subcellular distribution of the Ca2+, phospholipid-dependent protein kinase, protein kinase C (PKC). Using monoclonal antibodies that are specific for Type 3 (alpha) PKC, we have determined that there are least two pools of PKC in normal rat embryo fibroblasts (REF52 cells): diffuse cytoplasmic and fiber-associated. Extraction with chelators and detergent before fixing and staining removes the cytoplasmic PKC. The fiber-associated staining remains in these cytoskeleton preparations. The cytoskeleton Type 3 PKC staining closely resembles that of the focal contact protein vinculin and colocalizes with another focal contact protein, talin. Cytochalasin, but not colchicine, coordinately disrupts the staining pattern of vinculin and PKC. Activation of PKC by treatment with phorbol esters causes depolymerization of microfilaments and reorganization of vinculin staining. We propose that Type 3 PKC is a modulatory component of the focal contact and has a primary role in regulation of the association of microfilament bundles with the plasma membrane.

Binding of Salmonella minnesota R-form glycolipid mR595 to rat fibroblasts and its effect on cell metabolism and cell behaviour.

Trypsinized normal rat embryo fibroblasts and untrypsinized and trypsinized transformed rat fibroblasts have two orders of binding sites for bacterial glycolipid mR595. The high order sites fix 1--3 micrograms glycolipid mR595/10(5) cells and those of the low order fix about 6 microgram glycolipids mR595/10(6) cells. Ca++ is required for the low order glycolipid mR595 binding to be trypsinized but not to the untrypsinized transformed rat fibroblasts. The low order binding is temperature dependent with the transition temperature lying between 25 and 37 degrees C. Exogenously added ganglioside and glycoproteins contained in the fetal calf serum do not inhibit fixation of glycolipid mR595. Only beta-lipoprotein at high concentrations is slightly inhibitory. Glycolipid mR595 fixation to transformed fibroblast does not alter their morphology and appears to slightly improve cell attachment to substratum. Glycolipid mR595 fixation results in a lengthening of the S-phase of the cell cycle and a reduction in 2-deoxyglucose uptake. Uptake of inorganic phosphate is not affected. Inhibition of phospholipid synthesis is observed in mR595 fixed fibroblasts whereas synthesis of cell surface glycoproteins and the content of cellular gangliosides is not affected.

Microfilament bundles, lets protein and growth control in somatic cell hybrids

Hybrid cell lines between normal rat embryo fibroblasts and TA3B mouse tumour cells, or between TA3B and BI hamster sarcoma cells, have been examined for the expression of the cell surface large external-transformation-sensitive (LETS) protein and the organization of microfilament bundles. LETS protein was detected by lactoperoxidase-catalysed radioiodination and microfilament bundles were visualized by indirect immunofluorescence with antibodies directed against actin or myosin. Hybrids which exhibited normal growth control characteristics had high levels of LETS protein and extensive microfilament bundles. Neoplastic transformation appears to be suppressed in these hybrids. Hybrids which had the growth control characteristics typical of transformed cells had reduced or zero levels of LETS protein and few microfilament bundles. These results confirm previous studies on the expression of the transformed phenotype in these hybrids and demonstrate that there is a good correlation between normal growth control and the presence of LETS protein and microfilament bundles. However, the changes in cell surface LETS protein and in the organization of microfilament bundles often appeared to be quantitative reductions rather than all-or-none effects. The magnitude of the alterations in the levels of LETS protein and in the organization of microfilaments appeared to correlate with the range of transformed characteristics exhibited by the cells. One transformed hybrid in particular, selected for growth in agar, had some surface LETS protein, some microfilament bundles and retained density-dependent inhibition of growth.

Ruthenium red-positive surface layer, extracellular filamentous material and intercellular junctions in hybrids between tumour and normal cells: abundant gap junctions correlate with density-dependent inhibition of growth.

The cell surface and intercellular junctions of transformed and non-transformed hybrid cells, obtained from fusion of murine mammary adenocarcinoma cells (TA3B) and normal rat embryofibroblasts (REF), were compared with those of the parental cells, using ruthenium red (RR) staining of cells fixed in situ. An RR-positive layer of variable thickness was found on the surface of all cell types. Measurements of the thickness of this layer on the free surface of the cell cultures deomonstrated a significant difference between transformed and non-transformed cells. The thickness distribution of the RR layer was similar on the surface of REF and non-transformed hybrid cells, but there was significant variation among all the transformed cell lines. Extracellular filamentous RR-positive material, usually in direct contact with the cell surface, was present in confluent cultures of REF and non-transformed hybrid cells, but was absent in the transformed cell lines. Gap junctions were few in both parental cell lines and rare or absent in transformed hybrids; a large increase in number and size of gap junctions was found in non-transformed hybrids. Abundance of long gap junctions was correlated with density-dependent inhibition of growth, since non-transformed hybrids grew in single layers whereas both normal and tumour parental cell types, and transformed hybrids grew in multilayers. Tight junctions were frequently encountered in TA3B and transformed hybrid cells but were not seen in REF cells and only occasionally seen in non-transformed hybrids. Intermediate-type junctions occurred in all cells, but desmosomes were found only rarely in TA3B cells and never in the other cell types.

A study of cell surface constituents restricting Salmonella minnesota R form glycopid mR595 fixation to normal rat embryo fibroblasts.

The nature of the cell surface constituent(s) containing sialic acid that block fixation of bacterial glycolipid mR595 to normal rat fibroblasts was explored. Indirect evidence suggests that a trypsin-sensitive, high molecular weight glycoprotein, present in larger amounts on the surface of normal cells (as compared with transformed rat fibroblasts), may be involved as a blocking agent.

Actin-containing cables within anchorage-dependent rat embryo cells are dissociated by plasmin and trypsin

The distribution of intracellular actin has been examined by specific immunofluorescence in a series of normal and SV40-transformed cell lines of rat origin. A consistent correlation was found between the presence of large thick sheathes of actin-containing material and anchorage-dependent growth control. Anchorage-independent growth by these cells has been shown to be associated with the production and secretion of a plasminogen activator, and dependent upon the presence of the active protease plasmin. We have found that these phenomena can be linked as follows. First, treatment with plasmin, but not urokinase or plasminogen, reversibly removes the actin-containing cables from normal rat embryo fibroblasts, and similar results are obtained with trypsin. Thrombin and chymotrypsin are relatively ineffective in causing the disappearance of the cables. Second, sera such as dog or monkey, which permit high levels of plasmin formation and activity, support cell growth in semi-solid media better than sera in which plasminogen is activated poorly or that are plasminogen-deficient; concomitantly, cables disappear in the former but not in the latter sera. The addition of a plasmin inhibitor prevents the disappearance of actin-containing cables.

Induction of type C virus-related functions in normal rat embryo fibroblasts by treatment with 5-iododeoxyuridine.

AbstractSecondary cultures of rat embryo fibroblasts, derived from six different strains of normal laboratory and wild rats, were treated with 5‐iododeoxyuridine in an attempt to establish the presence of endogenous type C viruses in rat cells. All the cell lines tested responded with a transient appearance of RNA‐dependent DNA polymerase (RDP) activity which reached a peak 3 days after the beginning of treatment. However, no viral particles or rat gs‐antigen could be detected at this stage. In the cells derived from two highly inbred strains, a spontaneous second burst of RDP activity was observed after 10–12 days, and on subculturing these cells a 20‐ to 40‐fold increase of enzyme activity could be obtained. During this peak of activity both gs‐antigen and viral particles capable of incorporating labelled uridine were detected. Electron microscopic examination revealed the presence of complete or immature virions in one cell strain. The virus induced was non‐infective for monolayer cultures of 10 different animal species. Attempts to rescue infective virus by complementation with murine leukemia or sarcoma viruses failed. Prolonged cultivation of treated cultures did yield cells with trasformed morphology, but no oncogenicity could be demonstrated for these cells.

Fixation of a Salmonella minnesota R-form glycolipid on the membrane of normal and transformed rat-embryo fibroblasts.

Binding of Salmonella minnesota R form glycolipid mR595 to cultured rat embryo fibroblasts has been demonstrated by three methods: by uptake of labelled glycolipid, by passive hemagglutination and by immunofluorescence. The results by the three methods are correlative. Normal rat embryo fibroblasts exhibit lower fixation capacity than SV40 transformed rat embryo fibroblasts. Trypsinization of the two types of cells results in increased fixation of the bacterial glycolipid. Glycolipid mR595 binding is localized at the cell surface.