Isolation and Characterization of a Near-Haploid Human Cell Line

Kotecki, Maciej

doi:10.1006/excr.1999.4656

Cited by 140 publications

(136 citation statements)

References 27 publications

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“…To identify genetic factors that determine the sensitivity of TP53-mutant cancer cells to Wee1 inhibition, a loss-of-function genetic screen was performed in the near-haploid human KBM-7 cell line, which harbors a serine (Ser)-to-glutamine (Glu) missense mutation at Ser-215 in p53 (26). The near-haploid karyotype of the KBM-7 cells allows "gene-trap" retroviruses that carry a strong splice acceptor to randomly create instantaneous gene knockouts, a process referred to as insertional mutagenesis.…”

Section: Resultsmentioning

confidence: 99%

A haploid genetic screen identifies the G₁/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

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Bisteau

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The Wee1 cell cycle checkpoint kinase prevents premature mitotic entry by inhibiting cyclin-dependent kinases. Chemical inhibitors of Wee1 are currently being tested clinically as targeted anticancer drugs. Wee1 inhibition is thought to be preferentially cytotoxic in p53-defective cancer cells. However, TP53 mutant cancers do not respond consistently to Wee1 inhibitor treatment, indicating the existence of genetic determinants of Wee1 inhibitor sensitivity other than TP53 status. To optimally facilitate patient selection for Wee1 inhibition and uncover potential resistance mechanisms, identification of these currently unknown genes is necessary. The aim of this study was therefore to identify gene mutations that determine Wee1 inhibitor sensitivity. We performed a genome-wide unbiased functional genetic screen in TP53 mutant near-haploid KBM-7 cells using gene-trap insertional mutagenesis. Insertion site mapping of cells that survived long-term Wee1 inhibition revealed enrichment of G 1 /S regulatory genes, including SKP2, CUL1, and CDK2. Stable depletion of SKP2, CUL1, or CDK2 or chemical Cdk2 inhibition rescued the γ-H2AX induction and abrogation of G 2 phase as induced by Wee1 inhibition in breast and ovarian cancer cell lines. Remarkably, live cell imaging showed that depletion of SKP2, CUL1, or CDK2 did not rescue the Wee1 inhibition-induced karyokinesis and cytokinesis defects. These data indicate that the activity of the DNA replication machinery, beyond TP53 mutation status, determines Wee1 inhibitor sensitivity, and could serve as a selection criterion for Wee1-inhibitor eligible patients. Conversely, loss of the identified S-phase genes could serve as a mechanism of acquired resistance, which goes along with development of severe genomic instability.cell cycle | checkpoint | AZD-1775 | MK-1775 | polyploidy

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Section: Resultsmentioning

confidence: 99%

A haploid genetic screen identifies the G₁/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

Heijink

Blomen

Bisteau

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…S5). 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). In the future, it will be interesting to further define the requirements for maintaining a haploid genome in differentiated cells.…”

Section: Research Reportmentioning

confidence: 99%

Germline potential of parthenogenetic haploid mouse embryonic stem cells

et al. 2012

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SUMMARYHaploid 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.

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“…This is because a clearly distinguishable phenotype is evident, even in the case of recessive mutations of essential genes, due to the absence of an alternate gene copy to compensate for the modified or missing function. Therefore, researchers have endeavored to establish haploid cell lines from diploid organisms, such as human KBM-7 leukemia cell lines (Carette et al, 2009;Kotecki et al, 1999) and medaka ES cells (Yi et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Induction of the G2/M transition stabilizes haploid embryonic stem cells

et al. 2014

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The recent successful establishment of mouse parthenogenetic haploid embryonic stem cells ( phESCs) and androgenetic haploid ESCs (ahESCs) has stimulated genetic research not only in vitro but also in vivo because of the germline competence of these cell lines. However, it is difficult to maintain the haploid status over time without a frequent sorting of the G1 phase haploid ESCs by fluorescenceactivated cell sorting (FACS) at short intervals, because haploid cells tend to readily self-diploidize. To overcome this spontaneous diploid conversion, we developed a phESC culture condition using a small molecular inhibitor of Wee1 kinase to regulate the cell cycle by accelerating the G2/M phase transition and preventing re-entry into extra G1/S phase. Here, we demonstrate that, under this condition, phESCs maintained the haploid status for at least 4 weeks without the need for FACS. This method will greatly enhance the availability of these cells for genetic screening.

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Isolation and Characterization of a Near-Haploid Human Cell Line

Cited by 140 publications

References 27 publications

A haploid genetic screen identifies the G₁/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

A haploid genetic screen identifies the G₁/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

Germline potential of parthenogenetic haploid mouse embryonic stem cells

Induction of the G2/M transition stabilizes haploid embryonic stem cells

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Isolation and Characterization of a Near-Haploid Human Cell Line

Cited by 140 publications

References 27 publications

A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

Germline potential of parthenogenetic haploid mouse embryonic stem cells

Induction of the G2/M transition stabilizes haploid embryonic stem cells

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Product

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A haploid genetic screen identifies the G₁/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

A haploid genetic screen identifies the G₁/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity