Opposing roles for 53BP1 during homologous recombination

Although DNA non-homologous end-joining repairs most DNA double-strand breaks (DSBs) in G2 phase, late repairing DSBs undergo resection and repair by homologous recombination (HR). Based on parallels to the situation in G1 cells, previous work has suggested that DSBs that undergo repair by HR predominantly localize to regions of heterochromatin (HC). By using H3K9me3 and H4K20me3 to identify HC regions, we substantiate and extend previous evidence, suggesting that HC-DSBs undergo repair by HR. Next, we examine roles for 53BP1 and BRCA1 in this process. Previous studies have shown that 53BP1 is pro-non-homologous end-joining and anti-HR. Surprisingly, we demonstrate that in G2 phase, 53BP1 is required for HR at HC-DSBs with its role being to promote phosphorylated KAP-1 foci formation. BRCA1, in contrast, is dispensable for pKAP-1 foci formation but relieves the barrier caused by 53BP1. As 53BP1 is retained at irradiation-induced foci during HR, we propose that BRCA1 promotes displacement but retention of 53BP1 to allow resection and any necessary HC modifications to complete HR. In contrast to this role for 53BP1 in HR in G2 phase, we show that it is dispensable for HR in S phase, where HC regions are likely relaxed during replication

Long-Term Quiescent Fibroblast Cells Transit into Senescence

Cellular senescence is described to be a consequence of telomere erosion during the replicative life span of primary human cells. Quiescence should therefore not contribute to cellular aging but rather extend lifespan. Here we tested this hypothesis and demonstrate that cultured long-term quiescent human fibroblasts transit into senescence due to similar cellular mechanisms with similar dynamics and with a similar maximum life span as proliferating controls, even under physiological oxygen conditions. Both, long-term quiescent and senescent fibroblasts almost completely fail to undergo apoptosis. The transition of long-term quiescent fibroblasts into senescence is also independent of HES1 which protects short-term quiescent cells from becoming senescent. Most significantly, DNA damage accumulates during senescence as well as during long-term quiescence at physiological oxygen levels. We suggest that telomere-independent, potentially maintenance driven gradual induction of cellular senescence during quiescence is a counterbalance to tumor development

Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts.

Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD), fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P) via reversibly cell cycle arrested (C) to irreversibly arrested senescent cells (S). In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing

Effect of long term quiescence induction (100 or 150 days) in MRC-5 fibroblasts maintained at 20% O₂.

<p>(<b>A</b>) Growth curve of 3 independent MRC-5 fibroblast cell lines (control with no quiescence induction, and cell lines where quiescence was induced for 100 or 150 days respectively by contact inhibition and then maintained in culture till they approached senescence) maintained in culture at 20% O₂ as triplicates from an early PD until senescence at late PDs. Each growth curve is measured in triplicate. Data points of all measurements are displayed (not the mean). (<b>B & C</b>) Percentage of SA-β gal positive cells at different time points of their growth in culture in the control MRC-5 fibroblast cell line and in the cell lines where quiescence was induced for 100 or 150 days respectively. Fig. 2 B and C are plotted with PDs and days in the y-axis respectively. Each curve is measured in triplicate, the mean value is displayed with error bar (± S.E). (<b>D</b>) The blots show the protein expression levels of p16, p21, p27, Cyclin D1, Cyclin D2, Ki-67 and γH2A.X in MRC-5 fibroblast cell lines (subjected to different culture conditions of 100 or 150 days quiescence by contact inhibition and no quiescence induction) maintained in culture at 20% O₂ until they approached senescence at late PD. The up or down-regulation was signified by the presence or absence of the bands in Western Blots. (<b>E, F, G, H, I, J, K</b>) Comparison of mean fold change of protein expression levels of p16 (E), p21 (F), p27 (G), Cyclin D1 (H), Cyclin D2 (I), Ki-67 (J) and γH2A.X (K) in MRC-5 cell lines where quiescence was induced for 100 or 150 days by contact inhibition respectively compared to controls at corresponding span of time in culture. Cell lines were maintained at 20% O₂ as triplicates. The bars indicate the mean ± S.D. *** p<0.001 - significantly different compared to fibroblasts with PD assigned 1. n = 3.</p

Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer’s Aβ peptide

The oligomerization of A&beta; peptide into amyloid fibrils is a hallmark of Alzheimer&rsquo;s disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of A&beta; and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of A&beta; peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of A&beta; fibrillation. The three histidine residues at positions 6, 13 and 14 of A&beta;(1&ndash;40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged

Opposing roles for 53BP1 during homologous recombination

Although DNA non-homologous end-joining repairs most DNA double-strand breaks (DSBs) in G2 phase, late repairing DSBs undergo resection and repair by homologous recombination (HR). Based on parallels to the situation in G1 cells, previous work has suggested that DSBs that undergo repair by HR predominantly localize to regions of heterochromatin (HC). By using H3K9me3 and H4K20me3 to identify HC regions, we substantiate and extend previous evidence, suggesting that HC-DSBs undergo repair by HR. Next, we examine roles for 53BP1 and BRCA1 in this process. Previous studies have shown that 53BP1 is pro-non-homologous end-joining and anti-HR. Surprisingly, we demonstrate that in G2 phase, 53BP1 is required for HR at HC-DSBs with its role being to promote phosphorylated KAP-1 foci formation. BRCA1, in contrast, is dispensable for pKAP-1 foci formation but relieves the barrier caused by 53BP1. As 53BP1 is retained at irradiation-induced foci during HR, we propose that BRCA1 promotes displacement but retention of 53BP1 to allow resection and any necessary HC modifications to complete HR. In contrast to this role for 53BP1 in HR in G2 phase, we show that it is dispensable for HR in S phase, where HC regions are likely relaxed during replication

Fit of Eq. 1 to constant growth for HeLa (own data: green squares, fit: blue line) and rat fibroblast cells (data: blue circles [<b>53</b>], fit: red dashed line).

<p>See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042150#pone-0042150-t001" target="_blank">Table 1</a>.</p

Values of the parameter f₁ in the model extension.

<p>f₁ values for model extension with cell cycle arrest species. Same experimental data as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042150#pone-0042150-t001" target="_blank">Table 1</a>. For all simulations parameter r  =  r_max is set to 0.70 determined for HeLa cell growth rate and parameter f₂ to 1.</p

Simulation of WI-38 fibroblast data.

<p>a, Experimental growth data (circled) were fitted by model Eq. 3a–d using Eq. 4 as an expression for monotonically increasing stress γ; b, the fraction of proliferating cells P, cells showing a cell cycle arrest or a senescent phenotype (C+S) and solely the fraction of senescent cells S are shown together with the appearance of biomarkers. Biomarker values (p16, p21, SA-β-Gal and SAHF) were measured by immune-fluorescence as number of positive cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042150#pone.0042150-Klement1" target="_blank">[49]</a>.</p