agar and Fibrosarcoma

To investigate the cell biological function of PU.1, a member of the Ets family of transcription factors, a vector capable of expressing the protein was transfected into HT1080 human fibrosarcoma cells. Exogenous expression of PU.1 in HT1080 cells reduced colony-forming efficiency but stimulated cell migration in soft agar, although it did not affect cell growth in adherent culture. Expression of the urokinase-type plasminogen activator (uPA) mRNA, which is known to be correlated with cell migration and invasion, was enhanced in PU.1 transfectants compared with mock transfectants. Run-on analysis demonstrated that uPA transcription was unaffected by PU.1, suggesting that this enhancement mainly occurs at a post-transcriptional level. On the other hand, treatment of HT1080 cells with the synthetic glucocorticoid dexamethasone (DEX; 10(-7) M) significantly reduced uPA gene expression at a transcriptional level. Furthermore, DEX inhibited cell migration in soft agar without affecting cell growth. These negative effects of DEX on uPA expression and cell migration were alleviated by the expression of PU.1 in HT1080 cells, whereas expression of the N-ras oncogene, which is responsible for maintenance of the transformed phenotypes in HT1080 cells, was unaffected by PU.1 expression or DEX treatment in the cells. Our results suggest that expression of PU.1 can stimulate uPA gene expression at the post-transcriptional level, which may subsequently lead to activation of cell motility and/or reduced cell-cell adhesion, but reduces anchorage-independent growth of HT1080 cells.

Topics: Agar; Cell Movement; Dexamethasone; Fibrosarcoma; Gene Expression Regulation; Genes, ras; Glucocorticoids; Humans; Proto-Oncogene Proteins; Trans-Activators; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Tumor Stem Cell Assay; Urokinase-Type Plasminogen Activator

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed almost exclusively by glial cells of the central nervous system. We have previously transfected GFAP-negative human astrocytoma cells with the gene for GFAP and have demonstrated that GFAP transfection decreases astrocytoma proliferation and alters astrocytoma morphology. To determine if the same cellular responses could be elicited upon GFAP transfection of nonglial tumor cells, in the present study we have transfected a GFAP-negative human malignant fibrosarcoma cell line (HT-1080) with a cDNA containing the entire coding sequence of the human GFAP gene under the control of an inducible metallothionein promoter. Stably transfected HT-1080 clones were identified that are GFAP-positive by PCR and immunocytochemistry. GFAP-positive HT-1080 fibrosarcoma cells also demonstrate a decrease in tumor cell proliferation, altered morphological features characterized by cell elongation and cytoplasmic process formation, and reduction of invasive potential when compared to controls. These findings suggest that the inducible expression of the cytoskeletal protein GFAP can also be associated with dramatic cellular effects in nonglial non-central nervous system tumor cells.

Topics: Agar; Blotting, Western; Cell Division; Cloning, Molecular; Collagen; Drug Combinations; Extracellular Matrix; Fibrosarcoma; Gene Expression Regulation, Neoplastic; Genetic Testing; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Laminin; Metallothionein; Neoplasm Invasiveness; Promoter Regions, Genetic; Proteoglycans; Thymidine; Transfection; Tritium; Tumor Cells, Cultured

Topics: Agar; Animals; Cells, Cultured; Clone Cells; Culture Media; Female; Fibrosarcoma; Mice; Neoplasm Metastasis; Neoplasms, Experimental

The pattern of in vitro anchorage-independent growth of tumor cells from the murine UV-2237 fibrosarcoma correlated with their ability to produce experimental metastasis in vivo. When seeded into 0.3% Noble agar semisolid medium, cells of metastatic clones developed into larger tumor colonies at a faster rate than did cells of clones with low metastatic potential. Furthermore, when tumor cells were plated into 0.6% Noble agar, colony development by cells of low metastatic potential clones was almost completely restricted. Tumor cells from the heterogeneous parent UV-2237 fibrosarcoma were plated into dishes containing 0.6% agar semisolid medium. In separate experiments, 16 colonies were isolated 2 weeks thereafter and were established as individual cell lines in monolayer cultures. All of these cell lines produced experimental metastases as determined by in vivo lung colony assay. The data suggest that anchorage-independent growth of UV-2237 tumor cells in 0.6% Noble agar semisolid medium is selective and permits the isolation of metastatic subpopulations.

Topics: Agar; Animals; Cell Adhesion; Cell Division; Cells, Cultured; Culture Media; Fibrosarcoma; Lung Neoplasms; Mice; Neoplasm Metastasis; Sarcoma, Experimental

The radiation sensitivity of a weakly immunogenic spontaneous fibrosarcoma of the C3Hf/Sed mouse (designated FSa-II) was assessed by three in vivo cell survival methods: end-point dilution (TD50) assay, lung colony (LC) assay, and agar diffusion chamber (ADC) assay. The hypoxic fraction of this tumour was also determined by the ADC method. Although there was a good agreement of the cell survival data between the ADC and LC methods, the TD50 method yielded a considerably less steep cell survival curve. Beneficial aspects and limitations of each assay are discussed. In addition, the use of the ADC method for the growth of xenogeneic cell lines and a preliminary experiment with human tumour cells in non-immunosuppressed hosts suggest that this method may be a valuable adjunct for studying the growth and therapeutic responses of human tumour cells.

Topics: Agar; Animals; Biological Assay; Cell Survival; Clone Cells; Fibrosarcoma; Lung; Mice; Neoplasm Transplantation; Oxygen; Radiation Tolerance; Sarcoma, Experimental; Transplantation, Homologous

Topics: Agar; Animals; Benz(a)Anthracenes; Benzopyrans; Cell Count; Cell Transformation, Neoplastic; Chromosomes; Clone Cells; Cricetinae; Culture Media; Culture Techniques; Female; Fibrosarcoma; Karyotyping; Male; Methods; Mitosis; Neoplasm Transplantation; Neoplasms, Experimental; Polycyclic Compounds; Transplantation, Homologous