A rapid and optimization-free procedure allows the in vivo detection of subtle cell cycle and ploidy alterations in tissues by flow cytometry

Heinlein, Christina; Deppert, Wolfgang; Braithwaite, Antony W.; Speidel, Daniel

doi:10.4161/cc.9.17.12831

Cited by 24 publications

(24 citation statements)

References 25 publications

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“…Flow cytometry and cell sorting are regularly used to analyse and acquire specific cell populations allowing the study of cells carrying fluorescence emitting reporter genes for specific genes of interest. Significant improvement in the quality of DNA content analysis have been made; high resolution cell cycle profiles and ploidy alterations (DNA-indices as small as 1.09) can be now detected in tumour samples [43]. Flow karyotyping (analysis of chromosomes in suspension by flow-cytometry) can be used to detect chromosomal rearangements and specific repetitive DNA sequences [44].…”

Section: Discussionmentioning

confidence: 99%

Live cell detection of chromosome 2 deletion and Sfpi1/PU1 loss in radiation-induced mouse acute myeloid leukaemia

et al. 2013

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The CBA/H mouse model of radiation-induced acute myeloid leukaemia (rAML) has been studied for decades to bring to light the molecular mechanisms associated with multistage carcinogenesis. A specific interstitial deletion of chromosome 2 found in a high proportion of rAML is recognised as the initiating event. The deletion leads to the loss of Sfpi, a gene essential for haematopoietic development. Its product, the transcription factor PU.1 acts as a tumour suppressor in this model. Although the deletion can be detected early following ionising radiation exposure by cytogenetic techniques, precise characterisation of the haematopoietic cells carrying the deletion and the study of their fate in vivo cannot be achieved. Here, using a genetically engineered C57BL/6 mouse model expressing the GFP fluorescent molecule under the control of the Sfpi1 promoter, which we have bred onto the rAML-susceptible CBA/H strain, we demonstrate that GFP expression did not interfere with X-ray induced leukaemia incidence and that GFP fluorescence in live leukaemic cells is a surrogate marker of radiation-induced chromosome 2 deletions with or without point mutations on the remaining allele of the Sfpi1 gene. This study presents the first experimental evidence for the detection of this leukaemia initiating event in live leukemic cells.

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Section: Discussionmentioning

confidence: 99%

Live cell detection of chromosome 2 deletion and Sfpi1/PU1 loss in radiation-induced mouse acute myeloid leukaemia

et al. 2013

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“…Cell dispersal and fixation was performed on freshly dissected cerebellum as previously described with minor modifications [47,48] (additional 7 JJ and 9 jj Gunn pups). Briefly, small pieces of the tissue were mechanically dispersed by 100 µm and 40 µm nylon cell strainers (BD Falcon # 352560 and # 352540, BD Bioscience, Bedford, USA).…”

Section: Methodsmentioning

confidence: 99%

Alterations in the Cell Cycle in the Cerebellum of Hyperbilirubinemic Gunn Rat: A Possible Link with Apoptosis?

et al. 2013

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Severe hyperbilirubinemia causes neurological damage both in humans and rodents. The hyperbilirubinemic Gunn rat shows a marked cerebellar hypoplasia. More recently bilirubin ability to arrest the cell cycle progression in vascular smooth muscle, tumour cells, and, more importantly, cultured neurons has been demonstrated. However, the involvement of cell cycle perturbation in the development of cerebellar hypoplasia was never investigated before. We explored the effect of sustained spontaneous hyperbilirubinemia on cell cycle progression and apoptosis in whole cerebella dissected from 9 day old Gunn rat by Real Time PCR, Western blot and FACS analysis. The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001), characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05), cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively) and cyclin dependent kinases2 (25%, p < 0.001). This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001) which amplifies the apoptotic pathway. In line with this was the increase of the cleaved form of Poly (ADP-ribose) polymerase (54%, p < 0.01) and active Caspase3 (two fold, p < 0.01). These data indicate that the characteristic cerebellar alteration in this developing brain structure of the hyperbilirubinemic Gunn rat may be partly due to cell cycle perturbation and apoptosis related to the high bilirubin concentration in cerebellar tissue mainly affecting granular cells. These two phenomena might be intimately connected.

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“…Scale bar, 20 µm. (b) FACS analysis of cells isolated from normal lung, adenoma (a pool of eight tumors of 1–2 mm diameters), and adenomcarcinoma (tumors of 3 – 5 mm diameters), following a previously published protocol 51 . The data was processed using the FlowJo software.…”

Section: Figurementioning

confidence: 99%

Polyploid cells rewire DNA damage response networks to overcome replication stress-induced barriers for tumour progression

et al. 2012

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Mutations in genes involved in DNA replication such as FEN1, can cause single-stranded DNA breaks (SSBs) and subsequent collapse of DNA replication forks leading to DNA replication stresses. Persistent replication stresses normally induce p53-mediated senescence or apoptosis to prevent tumor progression. It is unclear how some mutant cells can overcome persistent replication stresses and bypass the p53-mediated pathways to develop malignancy. Here we show that formation of polyploidy, which is often observed in human cancers, leads to overexpression of BRCA1, p19arf and other DNA repair genes in FEN1 mutant cells. This overexpression triggers SSB repair and non-homologous end joining pathways to increase DNA repair activity, but at the cost of frequent chromosomal translocations. Meanwhile, DNA methylation silences p53 target genes, to bypass the p53-mediated senescence and apoptosis. These molecular changes rewire DNA damage response and repair gene networks in polyploid tumor cells, enabling them to escape replication stress-induced senescence barriers.

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A rapid and optimization-free procedure allows the in vivo detection of subtle cell cycle and ploidy alterations in tissues by flow cytometry

Cited by 24 publications

References 25 publications

Live cell detection of chromosome 2 deletion and Sfpi1/PU1 loss in radiation-induced mouse acute myeloid leukaemia

Live cell detection of chromosome 2 deletion and Sfpi1/PU1 loss in radiation-induced mouse acute myeloid leukaemia

Alterations in the Cell Cycle in the Cerebellum of Hyperbilirubinemic Gunn Rat: A Possible Link with Apoptosis?

Polyploid cells rewire DNA damage response networks to overcome replication stress-induced barriers for tumour progression

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