Partial reversion of the transformed phenotype in HRAS-transfected tumorigenic cells by transfer of a human gene (original) (raw)
Related papers
European Journal of Cell Biology, 1999
Differential display-ribosomal protein SIB-NADH-ubiquinone oxidoreductase-translational initiation factor 61p27 BBP-Kritl Chemical reverse transformation of CHO-KI and other cells is a well-established phenomenon, in which oncogenically transformed cells re-acquire fibroblastoid morphology, contact inhibition and anchorage-dependent growth, in response to cyclic AMP and other agents. A limited number of changes in gene transcription and enzyme activity have been demonstrated to coincide with these morphological and physiological changes. We have used a partial differential display to identify four genes that are transcriptionally modulated in reverse transformation. One of these, encoding ribosomal protein SIS, is transcriptionally suppressed, probably as a result of the detransforming process. Three others are transcriptionally activated. One has homology to NADH-ubiquinone oxidoreductase chain 4 protein, and is also probably changed as a result of the detransforming process. Another is homologous to a human sequence which encodes a 27 kDa protein, p27BBP/eIF6, that is involved in the biogenesis of 60S ribosomal subunit, and in cell lines of epithelial origin binds to fl integrin. This has not previously been described as transformation-related, and could have a causative role in reverse transformation. The third has homology, with transcriptional or processing variations, to a human genomic sequence, a positional candidate for a tumour suppressor gene, encoding the Kritl protein which interacts with the Ras-family GTPase Krev-I.
Transformation effector and suppressor genes
Journal of Cellular Biochemistry, 1991
Much has been learned about the molecular basis of cancer from the study of the dominantly acting viral and cellular oncogenes and their normal progenitors, the proto-oncogenes. More recent studies have resulted in the isolation and characterization of several genes prototypic of a second class of cancer genes. Whereas oncogenes act to promote the growth of cells, members of this latter class of genes act to inhibit cellular growth and are believed to contribute to the tumorigenic phenotype only when their activities are absent. This new class of cancer genes i s referred to by a number of different names including; anti-oncogenes, recessive oncogenes, growth suppressor genes, tumor suppressor genes and emerogenes. Although only a few of these cancer genes have been identified, to date, it is likely that many additional genes of this class await identification. A third class of genes, necessary for the development of the cancer phenotype, is comprised of the transformation effector genes. These are normal cellular genes that encode proteins that cooperate with or activate oncogene functions and thereby induce the development of the neoplastic phenotype. The inactivation of transformation effector functions would therefore inhibit the ability of certain dominantly acting oncogenes to transform cells. The approaches outlined here describe functional assays for the isolation and molecular characterization of transformation effector and suppressor genes.
Analysis of the Transforming Potential of the Human H-ras Gene by Random Mutagenesis
Proceedings of The National Academy of Sciences, 1984
Some tumor cells contain mutant ras genes that are capable of transforming NIH 3T3 cells. Those genes that have been analyzed arise from the wild-type, non-transforming ras genes by mutations producing single amino acid substitutions at position 12 or 61 of the encoded protein. We have performed random bisulfite-induced mutagenesis on the cloned wild-type human H-ras gene to find if mutations at other positions can activate the transforming potential of that gene. Most mutations are not activating, but mutations that specify single amino acid substitutions at position 12, 13, 59, or 63 of the encoded protein do activate the transforming potential of the H-ras gene. Some, but not all, mutant ras proteins show an altered electrophoretic mobility in NaDodSO4/polyacrylamide gels.
Molecular and cellular biology, 1986
We used mitotic chromosomes isolated from a human EJ bladder carcinoma cell line for morphological transformation of mouse C127 cells. These chromosome-mediated transformants were analyzed for cotransfer of markers syntenic with c-Ha-ras-1 on human chromosome 11. We also used cloned, dispersed human DNA repeats, in a general mapping strategy, to quantitate the amounts and molecular state of human DNA transferred along with the activated c-Ha-ras-1 gene. In situ hybridization was used to visualize the physical state of the transfected human chromatin. The combined use of these various techniques revealed the occurrence of both chromosomal and DNA rearrangements. However, our analysis also demonstrated that, in general, very substantial lengths of DNA are transferred intact. Closely linked markers are likely to cosegregate. Therefore, these transformants should be invaluable sources for the complete molecular cloning of isolated fragments of the short arm of human chromosome 11.
Reexpression of a cluster of silenced transgenes is associated with their rearrangement
Genes, Chromosomes and Cancer, 2001
Irreversible inactivation or silencing of tumor suppressor genes occurs frequently in the development of cancer. A similar process of silencing can occur after the integration of transfected or microinjected genes into the genomes of recipient cells. The inactivation of transfected genes seems particularly efficient in cells with stem cell characteristics. We have been studying the inactivation of genes transfected into cultured P19 embryonal carcinoma cells and found that the CpG-rich sequence comprising the coding region of the lacZ reporter gene becomes extensively methylated after integration into the genome. 5-Aza-2Ј-deoxycytidine (5AdC), an inhibitor of DNA methylation, induced the reexpression of silent transgenes in one clone of P19 cells studied in detail. However, the reexpressed genes remained heavily methylated over the lacZ coding sequence. We used pulsed-field gel electrophoresis to analyze the structure of the transgenic locus in the parental and in 5AdC-treated cells and found that, in each of the cells reexpressing the transgene, the cluster of transgenes had been rearranged. Each clone had undergone a different rearrangement that appeared to involve recombination within the tandemly repeated copies of the transgene. Our data seem consistent with the idea that 5AdC induces efficient DNA recombination between tandemly repeated genes and that the reexpression of silenced genes induced by 5AdC might be triggered by the chromatin reorganization at the site of DNA recombination.
Transformation of human cells by DNAs ineffective in transformation of NIH 3T3 cells
Proceedings of the …, 1985
Neonatal human foreskin fibroblasts can be transformed to anchorage-independent growth by transfection with DNAs inefficient in transforming NIH 3T3 cells. Human cells transfected with DNA from GM 1312, a multiple myeloma cell line, or MOLT-4, a permanent lymphoblast line, grow without anchorage at a much higher frequency than do the parental cells and their DNAs can transform human cell recipients to anchorage-independent growth; they have extended but not indefinite life spans and are nontumorigenic. Human fibroblasts are also transformed by DNAs from two multiple myeloma lines that also transform 3T3 cells; however, restriction analysis suggests that different transforming genes in this DNA are acting in the human and murine systems. These results indicate that the human cell transfection system allows detection of transforming genes not effective in the 3T3 system and points out the possibility of detection of additional transforming sequences even in DNAs that do transform murine cells.
GENETICS Materials and Methods
Tumor reversion is the process by which some cancer cells lose their malignant phenotype. This study was aimed at defining some of the molecular and phenotypic properties of this process. Biological models of tumor reversion were isolated from human leukemia and breast cancer cell lines by using the H-1 parvovirus as a selective agent. Differential gene expression analysis was performed between the parental malignant cells and their revertants or alternatively between these parental cells and their SIAH-1 transfectant counterparts. These SIAH-1 transfectants have a suppressed malignant phenotype and were used as a control for a viral-free system. Two hundred sixty-three genes were found to be either activated or inhibited during the reversion process, as confirmed by Northern blot analysis or quantitative PCR. Of these, 32% were differentially expressed in all systems, irrespective of whether parvovirus-selected, SIAH-1 overexpressing, or p53 mutant or wild-type cell lines were used, suggesting the existence of a universal mechanism underlying tumor reversion. Translationally Controlled Tumor Protein (tpt1͞TCTP) has the strongest differential expression, down-regulated in the reversion of U937-and SIAH-1-overexpressing cells. Inhibition of TCTP expression by anti-sense cDNA or small interfering RNA molecules results in suppression of the malignant phenotype and in cellular reorganization, similar to the effect of SIAH-1. Hence, tumor reversion can be defined at the molecular level, not just as the reversal of malignant transformation, but as a biological process in its own right involving a cellular reprogramming mechanism, overriding genetic changes in cancer, by triggering an alternative pathway leading to suppression of tumorigenicity.
Advances in Experimental Medicine and Biology, 2012
While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.