Checkpoints are blind to replication restart and recombination intermediates that result in gross chromosomal rearrangements

Replication fork inactivation can be overcome by homologous recombination, but this can cause gross chromosomal rearrangements that subsequently missegregate at mitosis, driving further chromosome instability. It is unclear when the chromosome rearrangements are generated and whether individual replication problems or the resulting recombination intermediates delay the cell cycle. Here we have investigated checkpoint activation during HR-dependent replication restart using a site-specific replication fork-arrest system. Analysis during a single cell cycle shows that HR-dependent replication intermediates arise in S phase, shortly after replication arrest, and are resolved into acentric and dicentric chromosomes in G2. Despite this, cells progress into mitosis without delay. Neither the DNA damage nor the intra-S phase checkpoints are activated in the first cell cycle, demonstrating that these checkpoints are blind to replication and recombination intermediates as well as to rearranged chromosomes. The dicentrics form anaphase bridges that subsequently break, inducing checkpoint activation in the second cell cycle

53BP1 can limit sister-chromatid rupture and rearrangements driven by a distinct ultrafine DNA bridging-breakage process

Chromosome missegregation acts as one of the driving forces for chromosome instability and cancer development. Here, we find that in human cancer cells, HeLa and U2OS, depletion of 53BP1 (p53-binding protein 1) exacerbates chromosome non-disjunction resulting from a new type of sister-chromatid intertwinement, which is distinct from FANCD2-associated ultrafine DNA bridges (UFBs) induced by replication stress. Importantly, the sister DNA intertwinements trigger gross chromosomal rearrangements through a distinct process, named sister-chromatid rupture and bridging. In contrast to conventional anaphase bridge-breakage models, we demonstrate that chromatid axes of the intertwined sister-chromatids rupture prior to the breakage of the DNA bridges. Consequently, the ruptured sister arms remain tethered and cause signature chromosome rearrangements, including whole-arm (Robertsonian-like) translocation/deletion and isochromosome formation. Therefore, our study reveals a hitherto unreported chromatid damage phenomenon mediated by sister DNA intertwinements that may help to explain the development of complex karyotypes in tumour cells

Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs

The DNA-dependent adenosine triphosphatase (ATPase) Plk1-interacting checkpoint helicase (PICH) has recently been implicated in spindle checkpoint (SAC) signaling (Baumann et al., Cell 128(1):101–114, 2007). Depletion of PICH by siRNA abolished the SAC and resulted in an apparently selective loss of Mad2 from kinetochores, suggesting a role for PICH in the regulation of the Mad1–Mad2 interaction. An apparent rescue of SAC functionality by overexpression of PICH in PICH-depleted cells initially seemed to confirm a role for PICH in the SAC. However, we have subsequently discovered that all PICH-directed siRNA oligonucleotides that abolish the SAC also reduce Mad2 mRNA and protein expression. This reduction is functionally significant, as PICH siRNA does not abolish SAC activity in a cell line that harbors a bacterial artificial chromosome driving the expression of murine Mad2. Moreover, we identified several siRNA duplexes that effectively deplete PICH but do not significantly affect SAC functionality or Mad2 abundance or localization. Finally, we discovered that the ability of overexpressed PICH to restore SAC activity in PICH-depleted cells depends on sequestration of the mitotic kinase Plk1 rather than ATPase activity of PICH, pointing to an underlying mechanism of “bypass suppression.” In support of this view, depletion or inhibition of Plk1 also rescued SAC activity in cells harboring low levels of Mad2. This observation suggests that a reduction of Plk1 activity partially compensates for reduced Mad2 levels and argues that Plk1 normally reduces the strength of SAC signaling. Collectively, our results question the role of PICH in the SAC and instead identify Mad2 as a sensitive off target for small RNA duplexes. In support of the latter conclusion, our evidence suggests that an off-target effect on Mad2 may also contribute to explain the apparent role of the Tao1 kinase in SAC signaling (Draviam et al., Nat Cell Biol 9(5):556–564, 2007)

PLK1 facilitates chromosome biorientation by suppressing centromere disintegration driven by BLM-mediated unwinding and spindle pulling

Centromeres provide a pivotal function for faithful chromosome segregation. They serve as a foundation for the assembly of the kinetochore complex and spindle connection, which is essential for chromosome biorientation. Cells lacking Polo-like kinase 1 (PLK1) activity suffer severe chromosome alignment defects, which is believed primarily due to unstable kinetochore-microtubule attachment. Here, we reveal a previously undescribed mechanism named ‘centromere disintegration’ that drives chromosome misalignment in PLK1-inactivated cells. We find that PLK1 inhibition does not necessarily compromise metaphase establishment, but instead its maintenance. We demonstrate that this is caused by unlawful unwinding of DNA by BLM helicase at a specific centromere domain underneath kinetochores. Under bipolar spindle pulling, the distorted centromeres are promptly decompacted into DNA threadlike molecules, leading to centromere rupture and whole-chromosome arm splitting. Consequently, chromosome alignment collapses. Our study unveils an unexpected role of PLK1 as a chromosome guardian to maintain centromere integrity for chromosome biorientation

Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality

Rescue of replication failure by Fanconi anaemia proteins

Chromosomal aberrations are often associated with incomplete genome duplication, for instance at common fragile sites, or as a consequence of chemical alterations in the DNA template that block replication forks. Studies of the cancer-prone disease Fanconi anaemia (FA) have provided important insights into the resolution of replication problems. The repair of interstrand DNA crosslinks induced by chemotherapy drugs is coupled with DNA replication and controlled by FA proteins. We discuss here the recent discovery of new FA-associated proteins and the development of new tractable repair systems that have dramatically improved our understanding of crosslink repair. We focus also on how FA proteins protect against replication failure in the context of fragile sites and on the identification of reactive metabolites that account for the development of Fanconi anaemia symptoms

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases

Systematic Review of Potential Health Risks Posed by Pharmaceutical, Occupational and Consumer Exposures to Metallic and Nanoscale Aluminum, Aluminum Oxides, Aluminum Hydroxide and Its Soluble Salts

Aluminum (Al) is a ubiquitous substance encountered both naturally (as the third most abundant element) and intentionally (used in water, foods, pharmaceuticals, and vaccines); it is also present in ambient and occupational airborne particulates. Existing data underscore the importance of Al physical and chemical forms in relation to its uptake, accumulation, and systemic bioavailability. The present review represents a systematic examination of the peer-reviewed literature on the adverse health effects of Al materials published since a previous critical evaluation compiled by Krewski et al. (2007). Challenges encountered in carrying out the present review reflected the experimental use of different physical and chemical Al forms, different routes of administration, and different target organs in relation to the magnitude, frequency, and duration of exposure. Wide variations in diet can result in Al intakes that are often higher than the World Health Organization provisional tolerable weekly intake (PTWI), which is based on studies with Al citrate. Comparing daily dietary Al exposures on the basis of “total Al”assumes that gastrointestinal bioavailability for all dietary Al forms is equivalent to that for Al citrate, an approach that requires validation. Current occupational exposure limits (OELs) for identical Al substances vary as much as 15-fold. The toxicity of different Al forms depends in large measure on their physical behavior and relative solubility in water. The toxicity of soluble Al forms depends upon the delivered dose of Al+ 3 to target tissues. Trivalent Al reacts with water to produce bidentate superoxide coordination spheres [Al(O2)(H2O4)+ 2 and Al(H2O)6 + 3] that after complexation with O2•−, generate Al superoxides [Al(O2•)](H2O5)]+ 2. Semireduced AlO2• radicals deplete mitochondrial Fe and promote generation of H2O2, O2 • − and OH•. Thus, it is the Al+ 3-induced formation of oxygen radicals that accounts for the oxidative damage that leads to intrinsic apoptosis. In contrast, the toxicity of the insoluble Al oxides depends primarily on their behavior as particulates. Aluminum has been held responsible for human morbidity and mortality, but there is no consistent and convincing evidence to associate the Al found in food and drinking water at the doses and chemical forms presently consumed by people living in North America and Western Europe with increased risk for Alzheimer\u27s disease (AD). Neither is there clear evidence to show use of Al-containing underarm antiperspirants or cosmetics increases the risk of AD or breast cancer. Metallic Al, its oxides, and common Al salts have not been shown to be either genotoxic or carcinogenic. Aluminum exposures during neonatal and pediatric parenteral nutrition (PN) can impair bone mineralization and delay neurological development. Adverse effects to vaccines with Al adjuvants have occurred; however, recent controlled trials found that the immunologic response to certain vaccines with Al adjuvants was no greater, and in some cases less than, that after identical vaccination without Al adjuvants. The scientific literature on the adverse health effects of Al is extensive. Health risk assessments for Al must take into account individual co-factors (e.g., age, renal function, diet, gastric pH). Conclusions from the current review point to the need for refinement of the PTWI, reduction of Al contamination in PN solutions, justification for routine addition of Al to vaccines, and harmonization of OELs for Al substances