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

Eukaryotic heterochromatin is characterized by a high density of repeats and transposons, as well as by modified histones, and influences both gene expression and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, we deleted the argonaute, dicer, and RNA-dependent RNA polymerase gene homologs, which encode part of the machinery responsible for RNA interference (RNAi). Deletion results in the aberrant accumulation of complementary transcripts from centromeric heterochromatic repeats. This is accompanied by transcriptional de-repression of transgenes integrated at the centromere, loss of histone H3 lysine-9 methylation, and impairment of centromere function. We propose that double-stranded RNA arising from centromeric repeats targets formation and maintenance of heterochromatin through RNAi.

http://science.sciencemag.org/content/sci/297/5588/1833.full.pdf

Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi.

Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin Attachment of sister kinetochores to the mitotic spindle during mitosis generates forces that would immediately split sister chromatids were it not opposed by cohesion Cohesion is essential for the alignment of chromosomes in metaphase but must be abolished for sister separation to start during anaphase In the budding yeast Saccharomyces cerevisiae, loss of sister-chromatid cohesion depends on a separating protein (separin) called Esp1 and is accompanied by dissociation from the chromosomes of the cohesion subunit Scc1 Here we show that Esp1 causes the dissociation of Scc1 from chromosomes by stimulating its cleavage by proteolysis A mutant Scc1 is described that is resistant to Esp1-dependent cleavage and which blocks both sister-chromatid separation and the dissociation of Scc1 from chromosomes The evolutionary conservation of separins indicates that the proteolytic cleavage of cohesion proteins might be a general mechanism for triggering anaphase

/pdf/sister-chromatid-separation-at-anaphase-onset-is-promoted-by-26yes098he.pdf

Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1

https://www.andrew.cmu.edu/course/03-541/papers/P1.pdf

Cleavage of Cohesin by the CD Clan Protease Separin Triggers Anaphase in Yeast

▪ Abstract The separation of sister chromatids at the metaphase to anaphase transition is one of the most dramatic of all cellular events and is a crucial aspect of all sexual and asexual reproduction. The molecular basis for this process has until recently remained obscure. New research has identified proteins that hold sisters together while they are aligned on the metaphase plate. It has also shed insight into the mechanisms that dissolve sister chromatid cohesion during both mitosis and meiosis. These findings promise to provide insights into defects in chromosome segregation that occur in cancer cells and into the pathological pathways by which aneuploidy arises during meiosis.

Disseminating the Genome: Joining, Resolving, and Separating Sister Chromatids During Mitosis and Meiosis

In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.

Dynamics of Centromeres during Metaphase–Anaphase Transition in Fission Yeast: Dis1 Is Implicated in Force Balance in Metaphase Bipolar Spindle

Cut1 is loaded onto the spindle by binding to Cut2 and promotes anaphase spindle movement upon Cut2 proteolysis

Background

The 20S cyclosome/APC complex promotes metaphase–anaphase transition by ubiquitinating its specific substrates such as mitotic cyclins and anaphase inhibitor Cut2/Pds1/securin. The complex has been shown to contain more than 10 proteins in budding yeast and frog. In fission yeast, however, only five (Cut4, Cut9, Nuc2, Apc10, Hcn1) have been identified.



Results

More than five hundred temperature-sensitive mutants were screened for identifying those defective in mitotic anaphase. Fifty-five showed the cut (cell untimely torn) phenotype or metaphase-arrest phenotypes, 27 of them locating at new loci. Their extracts were run in sucrose gradient centrifugation, and four showed alterations in the sedimentation profiles. The gene products of cut20+ and cut23+ were thus identified. Phenotypes of cut20–100 mutant highly resemble cut4–533 in many ways: they are hypersensitive to canavanine and CdCl2, and suppressed by PKA-inactivating regulators, cAMP-dependent phosphodiesterase and PKA regulatory subunits. Cut20 interacts closely with Cut4 in the assembly process of cyclosome. But cut20 mutant differs from cut4, as a novel gene stw1+ suppresses cut20 mutant but not cut4. cut23–194 mutant cells are sterile and blocked at metaphase, but does not show sensitivity to the stress and cAMP. TPR repeat-containing Cut23 may not be the stable component of APC/cyclosome, and its level significantly fluctuates during cell cycle. Cut23 may be ubiquitinated and degraded in a cell cycle dependent fashion.



Conclusions

We identified two new subunits of fission yeast cyclosome/APC complex. Our observations indicate that cyclosome components are divided into several subgroups with distinctly different roles.

Takashi Nakagawa

Papers

Dynamics of Centromeres during Metaphase–Anaphase Transition in Fission Yeast: Dis1 Is Implicated in Force Balance in Metaphase Bipolar Spindle

Cut1 is loaded onto the spindle by binding to Cut2 and promotes anaphase spindle movement upon Cut2 proteolysis

Fission yeast APC/cyclosome subunits, Cut20/Apc4 and Cut23/Apc8, in regulating metaphase-anaphase progression and cellular stress responses