Functional In Vitro Assays for the Isolation of Cell Transformation Effector and Suppressor Genes

• Published in: Environmental health perspectives Vol. 93; pp. 83 - 89
• Main Authors: Zarbl, Helmut, Kho, Choon-Joo, Boylan, Michael O., Van Amsterdam, John, Sullivan, Robert C., Hoemann, Caroline D., Afshani, Victoria L.
• Format: Journal Article
• Language: English
• Published: United States National Institute of Environmental Health Sciences. National Institutes of Health. Department of Health, Education and Welfare 01.06.1991
• Subjects: Animals; Cell Fusion; Cell growth; Cell Line, Transformed; Cell lines; Cell Separation; Cell Transformation, Neoplastic - genetics; Cell Transformation, Neoplastic - metabolism; Chromosome Mapping; Cloning, Molecular - methods; Fibroblasts; Flow Cytometry; Fluorescent Dyes - pharmacokinetics; Genes, Dominant; Genes, Retinoblastoma; Genes, Tumor Suppressor; Genetic mutation; Genetic Techniques; Mice; Mice, Nude; Mitochondria - metabolism; Neoplasms, Experimental - genetics; Oncogene Proteins v-fos - genetics; Oncogene Proteins v-fos - metabolism; Oncogenes; Phenotypes; Polymorphism, Restriction Fragment Length; Proto-Oncogenes; Rats; Rhodamine 123; Rhodamines - pharmacokinetics; Rodentia; Selection, Genetic; Somatic cells; Suppressor genes; Transfection; Transformed cell line; Tumor cell line; Tumor Suppressor Genes; Tumors
• ISSN: 0091-6765; 1552-9924
• DOI: 10.1289/ehp.919383

Malignant transformation may be viewed as an imbalance between signals inducing cell growth and signals leading to growth inhibition, differentiation, or senescence. A basic understanding of how these counterbalancing forces interact to regulate normal cell growth is the prerequisite to comprehending the mechanisms of tumorigenesis. Identification and characterization of the gene products implicated in these regulatory pathways is the first step toward understanding the disease process. The studies outlined here provide the potential basis for isolating and molecularly characterizing transformation effector and suppressor genes, which must respectively function in the positive and negative regulation of normal cell growth. The general strategy used involves the isolation and molecular characterization of nontransformed variants (revertants) from populations of tumor cells. The selection of revertants is facilitated by the ability to separate normal from transformed cells by fluorescence-activated sorting. The basis for this separation is the differential retention of the fluorescent dye rhodamine 123 in the mitochondria of normal versus transformed cells. Using this approach, we have isolated revertants from a mutagenized population of v-fos-transformed Rat-1 fibroblasts. Characterization of these clones indicated that they had sustained causal mutations in transformation effector genes. The unmutated effector genes are being identified and molecularly cloned by isolating retransformed clones from revertant cell lines that have been transfected with DNA or cDNA from normal primary cells. The same selection protocol has also been used to isolate revertants from tumor cell lines that have been transfected with DNA or cDNA from primary cells. The putative tumor-suppressor genes present in these revertants are currently being analyzed.