Many patients with locally advanced cervical cancer experience recurrence within the radiation fi... more Many patients with locally advanced cervical cancer experience recurrence within the radiation field after chemoradiotherapy. Biomarkers of tumor radioresistance are required to identify patients in need of intensified treatment. Here, the biomarker potential of miR‐200 family members was investigated in this disease. Also, involvement of tumor hypoxia in the radioresistance mechanism was determined, using a previously defined 6‐gene hypoxia classifier. miR‐200 expression was measured in pretreatment tumor biopsies of an explorative cohort (n = 90) and validation cohort 1 (n = 110) by RNA sequencing. Publicly available miR‐200 data of 79 patients were included for the validation of prognostic significance. A score based on expression of the miR‐200a/b/‐429 (miR‐200a, miR‐200b, and miR‐429) cluster showed prognostic significance in all cohorts. The score was significant in multivariate analysis of central pelvic recurrence. No association with distant recurrence or hypoxia status was found. Potential miRNA target genes were identified from gene expression profiles and showed enrichment of genes in extracellular matrix organization and cell adhesion. miR‐200a/b/‐429 overexpression had a pronounced radiosensitizing effect in tumor xenografts, whereas the effect was minor in vitro. In conclusion, miR‐200a/b/‐429 downregulation is a candidate biomarker of central pelvic recurrence and seems to predict cell adhesion‐mediated tumor radioresistance independent of clinical markers and hypoxia.
We discuss the mechanisms regulating entry into and progression through S phase in eukaryotic cel... more We discuss the mechanisms regulating entry into and progression through S phase in eukaryotic cells. Methods to study the G1/S transition are briefly reviewed and an overview of G1/S-checkpoints is given, with particular emphasis on fission yeast. Thereafter we discuss different aspects of the intra-S checkpoint and introduce the main molecular players and mechanisms.
GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein tra... more GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein translation in response to stress. Here, we show that GCN2 has an unexpected role in unstressed cells as a regulator of mitosis. This function is not through its canonical role in translation reprogramming, but through the regulation of two previously unidentified substrates, PP1α and γ. In the absence of GCN2 function, timing and levels of phosphorylation of key mitotic players are altered, leading to aberrant chromosome alignment, missegregating chromosomes, elevated number of tripolar spindles, and a delay in progression through mitosis. Pharmacological inhibition of GCN2 results in similar effects and is synergistic with Aurora A inhibition in causing more severe mitotic errors and cell death. We suggest that GCN2‐dependent phosphorylation of PP1α and γ restrains their activity and this is important to ensure the timely regulation of phosphorylation of several PP1 substrates during earl...
<p>A. Spot test for UVC sensitivity of wild-type (wt), <i>hpz1</i>Δ and <i&g... more <p>A. Spot test for UVC sensitivity of wild-type (wt), <i>hpz1</i>Δ and <i>rad26</i>Δ cells. Upper: unirradiated cells, center: 50J/m<sup>2</sup>, lower: 300J/m<sup>2</sup>. B: Survival after UVC irradiation of wild-type or <i>hpz1</i>Δ cells in G1, S or G2 phase. The survival of wild-type cells was normalized to 1 in each cell cycle phase (data without normalization are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044539#pone.0044539.s001" target="_blank">Fig. S1</a>). C. Survival of wild-type or <i>hpz1</i>Δ cells after HU-treatment. D. Microscopy images of <i>hpz1</i>Δ cells in HU (left) and 1 hour after release from HU (center) and wild-type cells released from HU (right). The DNA was stained with 4′,6-diamidino-2-phenylindole <b>(</b>DAPI). The arrows point to cut cells.</p
<p><b>A</b> The indicated strains were starved for leucine as described in Mate... more <p><b>A</b> The indicated strains were starved for leucine as described in Materials and Methods. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading. <b>B</b> eIF2α phosphorylation after UVC-irradiation in wild-type, <i>gcn2Δ</i> and <i>gcn1Δ</i> cells. Exponentially growing cells of the indicated strains were irradiated as described in Materials and methods and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182143#pone.0182143.ref023" target="_blank">23</a>]. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading. <b>C</b> preRC loading in <i>gcn1Δ</i> cells. Cells carrying a <i>cdc10-M17</i> mutation and GFP-tagged Mcm2 were grown in EMM medium, arrested in G1 by shifting them to 36°C for 4 h, released from the G1 block and irradiated with 1100 J/m<sup>2</sup> UVC. The percentage of cells containing chromatin-bound Mcm2:GFP was determined. The delay was calculated as the time difference between irradiated and unirradiated cells at reaching 70% of maximal preRC loading. <b>D</b> Prototroph wild-type cells were grown to mid-log phase in EMM medium. Supplements were added to 80 mg/l for 30 min before UV irradiation. Samples were taken immediately after irradiation. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading.</p
<p>Microscopy pictures of cells double-labelled with EdU and BrdU. Cells synchronized in G1... more <p>Microscopy pictures of cells double-labelled with EdU and BrdU. Cells synchronized in G1 phase were released and pulse-labelled in two consecutive S-phases, first with EdU, then with BrdU, as described in the text. Samples were harvested after the next mitosis, fixed and processed as described and imaged using fluorescence microscopy. Negative controls for cross-reactivity are provided on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088629#pone.0088629.s003" target="_blank">Fig. S3</a>.</p
Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources u... more Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources under steady-state conditions, with doubling times ranging from 2.5 to 14 hours. Flow cytometry and fluorescence microscopy confirmed earlier findings that at rapid growth rates, the G1 phase was short and cell separation occurred at the end of S phase. For some nitrogen sources, the growth rate was greatly decreased, the G1 phase occupied 30-50% of the cell cycle, and cell separation occurred in early G1. In contrast, other nitrogen sources supported low growth rates without any significant increase in G1 duration. The method described allows manipulation of the length of G1 and the relative cell cycle position of S phase in wild-type cells. Cell mass was measured by flow cytometry as scattered light and as protein-associated fluorescence. The extensions of G1 were not related to cell mass at entry into S phase. Our data do not support the hypothesis that the cells must reach a certain f...
Many patients with locally advanced cervical cancer experience recurrence within the radiation fi... more Many patients with locally advanced cervical cancer experience recurrence within the radiation field after chemoradiotherapy. Biomarkers of tumor radioresistance are required to identify patients in need of intensified treatment. Here, the biomarker potential of miR‐200 family members was investigated in this disease. Also, involvement of tumor hypoxia in the radioresistance mechanism was determined, using a previously defined 6‐gene hypoxia classifier. miR‐200 expression was measured in pretreatment tumor biopsies of an explorative cohort (n = 90) and validation cohort 1 (n = 110) by RNA sequencing. Publicly available miR‐200 data of 79 patients were included for the validation of prognostic significance. A score based on expression of the miR‐200a/b/‐429 (miR‐200a, miR‐200b, and miR‐429) cluster showed prognostic significance in all cohorts. The score was significant in multivariate analysis of central pelvic recurrence. No association with distant recurrence or hypoxia status was found. Potential miRNA target genes were identified from gene expression profiles and showed enrichment of genes in extracellular matrix organization and cell adhesion. miR‐200a/b/‐429 overexpression had a pronounced radiosensitizing effect in tumor xenografts, whereas the effect was minor in vitro. In conclusion, miR‐200a/b/‐429 downregulation is a candidate biomarker of central pelvic recurrence and seems to predict cell adhesion‐mediated tumor radioresistance independent of clinical markers and hypoxia.
We discuss the mechanisms regulating entry into and progression through S phase in eukaryotic cel... more We discuss the mechanisms regulating entry into and progression through S phase in eukaryotic cells. Methods to study the G1/S transition are briefly reviewed and an overview of G1/S-checkpoints is given, with particular emphasis on fission yeast. Thereafter we discuss different aspects of the intra-S checkpoint and introduce the main molecular players and mechanisms.
GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein tra... more GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein translation in response to stress. Here, we show that GCN2 has an unexpected role in unstressed cells as a regulator of mitosis. This function is not through its canonical role in translation reprogramming, but through the regulation of two previously unidentified substrates, PP1α and γ. In the absence of GCN2 function, timing and levels of phosphorylation of key mitotic players are altered, leading to aberrant chromosome alignment, missegregating chromosomes, elevated number of tripolar spindles, and a delay in progression through mitosis. Pharmacological inhibition of GCN2 results in similar effects and is synergistic with Aurora A inhibition in causing more severe mitotic errors and cell death. We suggest that GCN2‐dependent phosphorylation of PP1α and γ restrains their activity and this is important to ensure the timely regulation of phosphorylation of several PP1 substrates during earl...
<p>A. Spot test for UVC sensitivity of wild-type (wt), <i>hpz1</i>Δ and <i&g... more <p>A. Spot test for UVC sensitivity of wild-type (wt), <i>hpz1</i>Δ and <i>rad26</i>Δ cells. Upper: unirradiated cells, center: 50J/m<sup>2</sup>, lower: 300J/m<sup>2</sup>. B: Survival after UVC irradiation of wild-type or <i>hpz1</i>Δ cells in G1, S or G2 phase. The survival of wild-type cells was normalized to 1 in each cell cycle phase (data without normalization are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044539#pone.0044539.s001" target="_blank">Fig. S1</a>). C. Survival of wild-type or <i>hpz1</i>Δ cells after HU-treatment. D. Microscopy images of <i>hpz1</i>Δ cells in HU (left) and 1 hour after release from HU (center) and wild-type cells released from HU (right). The DNA was stained with 4′,6-diamidino-2-phenylindole <b>(</b>DAPI). The arrows point to cut cells.</p
<p><b>A</b> The indicated strains were starved for leucine as described in Mate... more <p><b>A</b> The indicated strains were starved for leucine as described in Materials and Methods. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading. <b>B</b> eIF2α phosphorylation after UVC-irradiation in wild-type, <i>gcn2Δ</i> and <i>gcn1Δ</i> cells. Exponentially growing cells of the indicated strains were irradiated as described in Materials and methods and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182143#pone.0182143.ref023" target="_blank">23</a>]. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading. <b>C</b> preRC loading in <i>gcn1Δ</i> cells. Cells carrying a <i>cdc10-M17</i> mutation and GFP-tagged Mcm2 were grown in EMM medium, arrested in G1 by shifting them to 36°C for 4 h, released from the G1 block and irradiated with 1100 J/m<sup>2</sup> UVC. The percentage of cells containing chromatin-bound Mcm2:GFP was determined. The delay was calculated as the time difference between irradiated and unirradiated cells at reaching 70% of maximal preRC loading. <b>D</b> Prototroph wild-type cells were grown to mid-log phase in EMM medium. Supplements were added to 80 mg/l for 30 min before UV irradiation. Samples were taken immediately after irradiation. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading.</p
<p>Microscopy pictures of cells double-labelled with EdU and BrdU. Cells synchronized in G1... more <p>Microscopy pictures of cells double-labelled with EdU and BrdU. Cells synchronized in G1 phase were released and pulse-labelled in two consecutive S-phases, first with EdU, then with BrdU, as described in the text. Samples were harvested after the next mitosis, fixed and processed as described and imaged using fluorescence microscopy. Negative controls for cross-reactivity are provided on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088629#pone.0088629.s003" target="_blank">Fig. S3</a>.</p
Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources u... more Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources under steady-state conditions, with doubling times ranging from 2.5 to 14 hours. Flow cytometry and fluorescence microscopy confirmed earlier findings that at rapid growth rates, the G1 phase was short and cell separation occurred at the end of S phase. For some nitrogen sources, the growth rate was greatly decreased, the G1 phase occupied 30-50% of the cell cycle, and cell separation occurred in early G1. In contrast, other nitrogen sources supported low growth rates without any significant increase in G1 duration. The method described allows manipulation of the length of G1 and the relative cell cycle position of S phase in wild-type cells. Cell mass was measured by flow cytometry as scattered light and as protein-associated fluorescence. The extensions of G1 were not related to cell mass at entry into S phase. Our data do not support the hypothesis that the cells must reach a certain f...
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