Isolation and Characterization of a Near-Haploid Human Cell Line
TL;DR: By serial subcloning, a near-haploid subclone (P1-55) is isolated from a heterogeneous human leukemia cell line, KBM-7, which has approximately half the human diploid DNA content and has a haploid karyotype except for a disomy of chromosome 8.
About: This article is published in Experimental Cell Research.The article was published on 1999-11-01. It has received 136 citations till now. The article focuses on the topics: Karyotype & Ploidy.
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TL;DR: New technologies can now provide unbiased quantification of multiple molecular and phenotypic changes across tens of thousands of individual cells from large numbers of perturbed cell populations simultaneously, which allow more accurate and comprehensive analyses of gene function in cells using genetic perturbation screens.
Abstract: Large-scale genetic perturbation screens are a classical approach in biology and have been crucial for many discoveries. New technologies can now provide unbiased quantification of multiple molecular and phenotypic changes across tens of thousands of individual cells from large numbers of perturbed cell populations simultaneously. In this Review, we describe how these developments have enabled the discovery of new principles of intracellular and intercellular organization, novel interpretations of genetic perturbation effects and the inference of novel functional genetic interactions. These advances now allow more accurate and comprehensive analyses of gene function in cells using genetic perturbation screens.
87 citations
TL;DR: The results suggest that in addition to being a peptide editor, TAPBPR improves peptide optimisation by promoting peptide-receptive MHC class I molecules to associate with the peptides-loading complex.
Abstract: Recently, we revealed that TAPBPR is a peptide exchange catalyst that is important for optimal peptide selection by MHC class I molecules. Here, we asked whether any other co-factors associate with TAPBPR, which would explain its effect on peptide selection. We identify an interaction between TAPBPR and UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1), a folding sensor in the calnexin/calreticulin quality control cycle that is known to regenerate the Glc1Man9GlcNAc2 moiety on glycoproteins. Our results suggest the formation of a multimeric complex, dependent on a conserved cysteine at position 94 in TAPBPR, in which TAPBPR promotes the association of UGT1 with peptide-receptive MHC class I molecules. We reveal that the interaction between TAPBPR and UGT1 facilities the reglucosylation of the glycan on MHC class I molecules, promoting their recognition by calreticulin. Our results suggest that in addition to being a peptide editor, TAPBPR improves peptide optimisation by promoting peptide-receptive MHC class I molecules to associate with the peptide-loading complex.
87 citations
Cites background from "Isolation and Characterization of a..."
...DOI: 10.7554/eLife.23049 3 of 25 UGT1 was observed in both IFN-g-treated HeLaM and KBM-7 cells (Figure 1A and B respectively)....
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...Although cell line authentication using short tandem repeat profiling was not undertaken for this work, the authenticity of HeLaM and KBM-7 cells was verified by the continu- ous confirmation that these cell lines had the expected HLA class I tissue type monitored by both staining with specific HLA antibodies and by mass spectrometry....
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...To induce/up-regulate TAPBPR expression and other components of the MHC class I antigen processing and presentation pathway, HeLaM and KBM-7 cells were treated with 100 U/ml IFN-g (Peprotech, UK) where indicated for 48– 72 h. Neerincx et al. eLife 2017;6:e23049....
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...…maintained in Dulbecco’s Modified Eagle’s medium (DMEM; Sigma-Aldrich, UK), while the near-haploid human chronic myeloid leukaemia cell line KBM-7 (Kotecki et al., 1999) and its variants (Duncan et al., 2012) (kind gifts from Lidia Duncan and Paul Lehner, University of Cambridge, UK) were…...
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...There was also no evidence of a disulphide-linked dimer between endoge- nously expressed TAPBPR and UGT1 in IFN-g-induced KBM-7 cells (Figure 3C)....
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TL;DR: By increasing the contribution of one-carbon units to the SAM from folate stores while decreasing SAH in response to AMPK-sensed energetic crisis, metformin can operate as a metabolo-epigenetic regulator capable of reprogramming one of the key conduits linking cellular metabolism to the DNA methylation machinery.
Abstract: The anti-diabetic biguanide metformin may exert health-promoting effects via metabolic regulation of the epigenome. Here we show that metformin promotes global DNA methylation in non-cancerous, cancer-prone and metastatic cancer cells by decreasing S-adenosylhomocysteine (SAH), a strong feedback inhibitor of S-adenosylmethionine (SAM)-dependent DNA methyltransferases, while promoting the accumulation of SAM, the universal methyl donor for cellular methylation. Using metformin and a mitochondria/complex I (mCI)-targeted analog of metformin (norMitoMet) in experimental pairs of wild-type and AMP-activated protein kinase (AMPK)-, serine hydroxymethyltransferase 2 (SHMT2)- and mCI-null cells, we provide evidence that metformin increases the SAM:SAH ratio-related methylation capacity by targeting the coupling between serine mitochondrial one-carbon flux and CI activity. By increasing the contribution of one-carbon units to the SAM from folate stores while decreasing SAH in response to AMPK-sensed energetic crisis, metformin can operate as a metabolo-epigenetic regulator capable of reprogramming one of the key conduits linking cellular metabolism to the DNA methylation machinery.
79 citations
TL;DR: It is shown that parthenogenetic haploids ESCs at high passage have robust germline competence enabling the production of transgenic mouse strains from genetically modified haploid ESCs and that a haploid karyotype is maintained when differentiation to an extra-embryonic fate is forced by induction of Gata6.
Abstract: Haploid embryonic stem cells (ESCs) have recently been derived from parthenogenetic mouse embryos and offer new possibilities for genetic screens. The ability of haploid ESCs to give rise to a wide range of differentiated cell types in the embryo and in vitro has been demonstrated. However, it has remained unclear whether haploid ESCs can contribute to the germline. Here, we show that parthenogenetic haploid ESCs at high passage have robust germline competence enabling the production of transgenic mouse strains from genetically modified haploid ESCs. We also show that differentiation of haploid ESCs in the embryo correlates with the gain of a diploid karyotype and that diploidisation is the result of endoreduplication and not cell fusion. By contrast, we find that a haploid karyotype is maintained when differentiation to an extra-embryonic fate is forced by induction of Gata6.
75 citations
Cites background from "Isolation and Characterization of a..."
...Interestingly, the proliferation of near-haploid human tumour cells might suggest that this requirement could be overcome in certain differentiated lineages through oncogenic signals (Kotecki et al., 1999; Carette et al., 2009; Carette et al., 2011)....
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TL;DR: Recently developed functional genomics technologies that address this need are reviewed and the approaches pioneered in model organisms, particularly in yeast, and more recently adapted to mammalian systems are discussed.
Abstract: The upswing in US Food and Drug Administration and European Medicines Agency drug approvals in 2014 may have marked an end to the dry spell that has troubled the pharmaceutical industry over the past decade. Regardless, the attrition rate of drugs in late clinical phases remains high, and a lack of target validation has been highlighted as an explanation. This has led to a resurgence in appreciation of phenotypic drug screens, as these may be more likely to yield compounds with relevant modes of action. However, cell-based screening approaches do not directly reveal cellular targets, and hence target deconvolution and a detailed understanding of drug action are needed for efficient lead optimization and biomarker development. Here, recently developed functional genomics technologies that address this need are reviewed. The approaches pioneered in model organisms, particularly in yeast, and more recently adapted to mammalian systems are discussed. Finally, areas of particular interest and directions for future tool development are highlighted.
72 citations
References
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TL;DR: The current status of gene targeting with particular emphasis on germ line modification of the mouse genome is discussed, and the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred are described.
Abstract: Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.
2,320 citations
TL;DR: Clones of homozygous fish have been produced from individual homozygotes and associated genetic methods facilitate genetic analyses of this vertebrate.
Abstract: Homozygous diploid zebra fish have been produced on a large scale by the application of simple physical treatments. Clones of homozygous fish have been produced from individual homozygotes. These clones and associated genetic methods will facilitate genetic analyses of this vertebrate.
1,203 citations
TL;DR: At the checkpoint between the prereplicative phase of growth and the phase of chromosome replication, cells lacking p21 failed to arrest the cell cycle in response to DNA damage, but their apoptotic response and genomic stability were unaltered.
Abstract: Most somatic cells die after a finite number of cell divisions, a phenomenon described as senescence. The p21 CIP1/WAF1 gene encodes an inhibitor of cyclin-dependent kinases. Inactivation of p21 by two sequential rounds of targeted homologous recombination was sufficient to bypass senescence in normal diploid human fibroblasts. At the checkpoint between the prereplicative phase of growth and the phase of chromosome replication, cells lacking p21 failed to arrest the cell cycle in response to DNA damage, but their apoptotic response and genomic stability were unaltered. These results establish the feasibility of using gene targeting for genetic studies of normal human cells.
827 citations
Journal Article•
TL;DR: The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.
Abstract: Rat fibroblast cell lines with targeted disruptions of both c-myc gene copies were constructed. Although c-myc null cells are viable, their growth is significantly impaired. The absence of detectable N-myc or L-myc expression indicates that Myc function is not absolutely essential for cell viability. The c-myc null phenotype is stable and can be reverted by introduction of a c-myc transgene. Exponentially growing c-myc null cells have the same cell size, rRNA, and total protein content as their c-myc +1+ parents, but the rates of RNA and protein accumulation as well as protein degradation are reduced. Both the G1 and G2 phases of the cell cycle are significantly lengthened, whereas the duration of S phase is unaffected. This is the first direct demonstration of a requirement for c-myc in G2. The G0-+S transition is synchronous, but S-phase entry is significantly delayed. The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.
473 citations
459 citations