During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes1,2. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants3,4,5, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization6, the selection of rare, spontaneously arising immortalized cells7,8,9,10, or the use of an entire viral genome11. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT)12 in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.

Creation of human tumour cells with defined genetic elements

Mitochondrial morphology is determined by a dynamic equilibrium between organelle fusion and fission, but the significance of these processes in vertebrates is unknown. The mitofusins, Mfn1 and Mfn2, have been shown to affect mitochondrial morphology when overexpressed. We find that mice deficient in either Mfn1 or Mfn2 die in midgestation. However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal. Embryonic fibroblasts lacking Mfn1 or Mfn2 display distinct types of fragmented mitochondria, a phenotype we determine to be due to a severe reduction in mitochondrial fusion. Moreover, we find that Mfn1 and Mfn2 form homotypic and heterotypic complexes and show, by rescue of mutant cells, that the homotypic complexes are functional for fusion. We conclude that Mfn1 and Mfn2 have both redundant and distinct functions and act in three separate molecular complexes to promote mitochondrial fusion. Strikingly, a subset of mitochondria in mutant cells lose membrane potential. Therefore, mitochondrial fusion is essential for embryonic development, and by enabling cooperation between mitochondria, has protective effects on the mitochondrial population.

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Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development

Stem cell factor is encoded at the SI locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor

https://www.cell.com/cell/pdf/0092-8674(88)90559-4.pdf

SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene

The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element.

The thermolabile large T antigen, encoded by the simian virus 40 early-region mutant tsA58, was used to establish clonal cell lines derived from rat embryo fibroblasts. These cell lines grew continuously at the permissive temperature but upon shift-up to the nonpermissive temperature showed rapidly arrested growth. The growth arrest occurred in either the G1 or G2 phase of the cell cycle. After growth arrest, the cells remained metabolically active as assayed by general protein synthesis and the ability to exclude trypan blue. The inability of these cell lines to divide at the nonpermissive temperature was not readily complemented by the exogenous introduction of other nuclear oncogenes. This finding suggests that either these genes establish cells via different pathways or that immortalization by one oncogene results in a finely balanced cellular state which cannot be adequately complemented by another establishment gene.

Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

Recombinant retroviruses that transduce the simian virus 40 (SV40) large T antigen or the polyomavirus large T antigen as well as encoding resistance to antibiotic G418 were used to investigate whether these genes alone were sufficient for immortalization of primary cells. The results provided definitive evidence that either viral gene can efficiently establish primary fibroblasts. The capability of the SV40 large T antigen to establish primary fibroblasts was undiminished by a mutation that alters its binding to sequences within the origin of replication. Surprisingly, most of the primary cells established by the expression of the SV40 large T antigen did not have a transformed phenotype. This suggests that transformation by SV40 is not simply due to a high level of expression of the SV40 large T antigen and stabilization of cellular p53.

Large T antigens of simian virus 40 and polyomavirus efficiently establish primary fibroblasts.

LargeT Antigens ofSimian Virus 40andPolyomavirus Efficiently Establish Primary Fibroblasts

The immunoglobulin heavy-chain enhancer is a cis-acting element which activates transcription of nearby genes only in cells of the lymphoid lineage. To identify the minimal sequences necessary to impart cell type transcriptional specificity, we tested the activity of several deletions and internal mutations in the mu enhancer. Experiments involving measurement of both chloramphenicol acetyltransferase activity and RNA levels indicated the presence of a dominant repressor element within the mu enhancer. This repressive activity was detected in fibroblasts but not in myeloma cells. Removal or disruption of this repressor element revealed the presence of elements within the mu enhancer that activate transcription in fibroblasts. Thus, enhancer tissue specificity is in part due to the composite of both constitutive activation and cell-type-specific repressive activity. The possible biological roles of this phenomenon are discussed.

P S Jat

Papers

Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

Large T antigens of simian virus 40 and polyomavirus efficiently establish primary fibroblasts.

LargeT Antigens ofSimian Virus 40andPolyomavirus Efficiently Establish Primary Fibroblasts

Localization of a repressive sequence contributing to B-cell specificity in the immunoglobulin heavy-chain enhancer.