The organizer of chromatin topology RIF1 ensures cellular resilience to DNA replication stress.

Eukaryotic genomes are duplicated from thousands of replication origins that fire sequentially forming a defined spatiotemporal pattern of replication clusters. The temporal order of DNA replication is determined by chromatin architecture and, more specifically, by chromatin contacts that are stabilized by RIF1. Here, we show that RIF1 localizes near newly synthesized DNA. In cells exposed to the DNA replication inhibitor aphidicolin, suppression of RIF1 markedly decreased the efficacy of isolation of proteins on nascent DNA, suggesting that the isolation of proteins on nascent DNA procedure is biased by chromatin topology. RIF1 was required to limit the accumulation of DNA lesions induced by aphidicolin treatment and promoted the recruitment of cohesins in the vicinity of nascent DNA. Collectively, the data suggest that the stabilization of chromatin topology by RIF1 limits replication-associated genomic instability.

Improving Patients' Life Quality after Radiotherapy Treatment by Predicting Late Toxicities.

Personalized treatment and precision medicine have become the new standard of care in oncology and radiotherapy. Because treatment outcomes have considerably improved over the last few years, permanent side-effects are becoming an increasingly significant issue for cancer survivors. Five to ten percent of patients will develop severe late toxicity after radiotherapy. Identifying these patients before treatment start would allow for treatment adaptation to minimize definitive side effects that could impair their long-term quality of life. Over the last decades, several tests and biomarkers have been developed to identify these patients. However, out of these, only the Radiation-Induced Lymphocyte Apoptosis (RILA) assay has been prospectively validated in multi-center cohorts. This test, based on a simple blood draught, has been shown to be correlated with late radiation-induced toxicity in breast, prostate, cervical and head and neck cancer. It could therefore greatly improve decision making in precision radiation oncology. This literature review summarizes the development and bases of this assay, as well as its clinical results and compares its results to the other available assays.

Imaging Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of oxaliplatin derivatives in human tissue sections.

Mass Spectrometry Imaging is an effective technology that allows to determine the in-situ distribution of endogen and/or exogen small molecules. It is a rapidly emerging approach for visualizing drugs and their metabolites within biological tissues. Matrix-Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry Imaging (MSI) coupled to high resolving power analyzer (e.g. TOF) was already investigated for metallodrug localization and metabolization studies, but was proved to suffer from a lack of sensitivity and resolution, leading to poor coverage and assignment. To counter these technological limitations, the use of ultra-high resolving power analyzer such as Fourier Transform Ion Cyclotron Resonance (FTICR) could be revealed as a technique of choice. The high field FTICR MS provides ultra-high resolving power and mass accuracy that allows exhaustive molecule coverage and non-ambiguous molecular formula assignments. Platinum derivatives, such as oxaliplatin, are widely used as therapeutic agents for cancer treatment. The assessment of their intake, distribution and metabolism within the organs is important to know the risks associated with their use. In this study, MALDI FTICR MSI analyses were performed to better understand the penetration and metabolization of platinum derivatives in ovaries of women treated by Hyperthermic Intraperitoneal Chemotherapy (HIPEC) for peritoneal metastasis of colorectal or appendicular origin. Twelve ovary sections, from six ovary samples in six women donors, before and after treatment, were analyzed with 120 µm spatial resolution. For the first time, the high resolving power (220,000 at m/z 457) and sub-ppm accuracy (<1 ppm) of the FTICR combined with an Isotopic Fine Structure study enabled to distinguish two Pt-isobaric species derived from oxaliplatin in biological tissues. One of these, which is unknown, was specifically localized at the contour of the ovary.

Evaluation of the environmental contamination and exposure risk in medical/non-medical staff after oxaliplatin-based pressurized intraperitoneal aerosol chemotherapy.

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a technique to directly deliver chemotherapeutic drugs in the abdomen for the treatment of peritoneal metastases. Pressurization improves the treatment efficacy but increases the risk of exposure for the medical/non-medical staff who can be exposed by dermal or ocular contact, or inhalation of aerosols containing the cytotoxic drugs. The aim of this study was to evaluate the risk of exposure for the medical/non-medical staff (nurses, surgeons, anaesthesiologists and cleaning personnel; n = 13) during PIPAC with oxaliplatin performed according to the protocol recommended in France. Blood samples were collected 1 h before and immediately after PIPAC, and urine samples 1 h before, and then 3 h and the morning after PIPAC. In the control, non-exposed group (n = 7), only one urine and blood sample were collected. Surface contamination in the operating room was assessed in water- and Surfanios-impregnated wipe samples. The total elemental platinum in each sample was quantified by inductively coupled plasma mass spectrometry, using a method adapted to quantify trace amounts (ng.L-1) in very low volumes (100 µl). No surface contamination was detected. Although 25% of urine samples in the exposed group contained platinum, no statistical difference was observed in urine and plasma samples collected before and after PIPAC and with the control group samples. These findings suggest that the French PIPAC protocol does not increase the risk of exposure to platinum in all staff categories involved. This protocol could be considered in future occupational policies and consensus statements. Trial registration: NCT04014426.

Study of oxaliplatin penetration into ovaries of patients treated with hyperthermic intraperitoneal chemotherapy (HIPEC) for peritoneal metastases of colorectal and appendiceal origin using mass spectrometry imaging.

OBJECTIVES: Platinum salts are commonly used in hyperthermic intraperitoneal chemotherapy (HIPEC) for digestive tract cancer treatment. During HIPEC with oxaliplatin for peritoneal metastases (PMs) treatment, the ovaries are directly exposed to the drug, questioning about ovarian resection and the potential impact of the drug on ovarian functionality, especially in young women of childbearing age. The goal of this work is to understand unwanted damages to the ovaries during HIPEC therapy by the determination of the concentration and distribution of platinum in ovaries in order to address its potential toxicity. METHODS: Mass spectrometry imaging techniques, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS), were used to study the penetration of oxaliplatin in ovaries after HIPEC treatment. RESULTS: MALDI-MS allowed the localization of an oxaliplatin-derivative (m/z 456.2) at the periphery of the ovaries. The quantitative LA-ICP MS maps confirmed the localization of elemental platinum as well as in the central part of ovaries from patients who received a previous platinum salt-based chemotherapy. CONCLUSIONS: LA-ICP MS images showed that platinum diffusion was extended in cases of previous systemic treatment, questioning about platinum derivatives gonado-toxicity when combining the two treatments.

Toward a comprehensive study for multielemental quantitative LA-ICP MS bioimaging in soft tissues.

Quantitative localization of metals in biological tissue sections is critical to obtain insight into metal toxicity mechanisms or their beneficial characteristics. This study presents the development of a quantitative LA-ICP MS bioimaging methodology based on the polymer film strategy and internal standardization. To maximize the number of elements mapped, an aqueous soluble polymer (dextran) was selected. Among the elements studied, the great majority (eight out eleven), i.e., Co, Ni, Cu, Zn, Se, Mo, Cd and Pt, exhibited linear regression after LA-ICP MS analysis of metal-spiked polymer standards. Methodology performances were carefully assessed as a function of the three internal standards (In, Rh and Ir) considered, the analytical operational conditions (ICP power, addition of O2 to ICP, and laser fluency) and the thickness of the biological tissue section. The results indicated that three groups (Co, Mo; Ni, Cu, Pt; and Zn, Se, Cd) of elements could be distinguished from their analytical response as a function of analytical conditions and the internal standard. These different element behaviors appeared to be mainly First Ionization Potential dependent (FIP). For elements with lower FIP (Co, Ni, Cu, Mo and Pt), differential responses due to carbon load in the ICP MS plasma could be efficiently corrected as a function of analytical conditions. Matrix effects were more pronounced for higher FIP elements (i.e., Zn, Cd and Se), and analysis of <10-µm thin sections without the addition of O2 to ICP MS plasma is recommended. LODs are in the range of 0.1-0.5 µg g-1 for Co, Mo, Cu, Ni, Pt and Cd as well as 0.9 and 1 µg g-1 for Zn and Se, respectively. The methodology was validated by means of a homemade metal-spiked kidney homogenate analyzed by LA-ICP MS imaging, and Co, Ni, Cu, Mo, and Pt provided the closest concentrations (5-29% bias) to the target values determined by ICP MS after mineralization. The methodology was applied to two types of clinical human samples undergoing different sample preparation protocols that did not affect internal standard homogeneity in the polymer film. This methodology is the first reported for the quantitative bioimaging of eight elements simultaneously.

An in-line enzymatic microreactor for the middle-up analysis of monoclonal antibodies by capillary electrophoresis.

Monoclonal antibodies (mAbs) are undergoing rapid growth in the pharmaceutical industry due to their clinical efficiency. Concomitantly, robust, cost-effective, and high throughput analytical methods are needed for their quality control. Among all analytical techniques, capillary electrophoresis (CE) presents alternative and attractive features because the capillary can be used both as a microreactor and as a support for separation. Transverse diffusion of laminar flow profiles was applied for the middle-up analysis of mAbs for the first time. Infliximab was selected as the model mAb. All middle-up analysis steps (enzymatic digestion, electrophoretic separation and UV detection) were integrated into the same capillary. The conditions for the separation of infliximab subunits (pH, ionic strength, and type of background electrolyte) and in-line digestion parameters (reactant injection conditions, time, temperature and enzyme/mAb ratio) were optimized. The in-line methodology was compared to the off-line methodology and evaluated in terms of proteolysis efficiency, repeatability, and applicability to different mAbs. Finally, the methodology was transferred to capillary electrophoresis coupled to mass spectrometry (sheathless interface) to identify infliximab subunits. The in-line methodology was successfully implemented with a simplified injection scheme, temperature control, fast enzymatic reaction and high resolution of separation of infliximab subunits under pseudo-native MS compatible conditions. In comparison with the off-line methodology, reactant consumption was reduced by a factor of 1000, and the numbers of theoretical plates were increased by a factor of 2.

Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites.

Imbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of dihydropyrimidines induces the formation of DNA-protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication invitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromosomal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.

Recruitment of ubiquitin-activating enzyme UBA1 to DNA by poly(ADP-ribose) promotes ATR signalling.

The DNA damage response (DDR) ensures cellular adaptation to genotoxic insults. In the crowded environment of the nucleus, the assembly of productive DDR complexes requires multiple protein modifications. How the apical E1 ubiquitin activation enzyme UBA1 integrates spatially and temporally in the DDR remains elusive. Using a human cell-free system, we show that poly(ADP-ribose) polymerase 1 promotes the recruitment of UBA1 to DNA. We find that the association of UBA1 with poly(ADP-ribosyl)ated protein-DNA complexes is necessary for the phosphorylation replication protein A and checkpoint kinase 1 by the serine/threonine protein kinase ataxia-telangiectasia and RAD3-related, a prototypal response to DNA damage. UBA1 interacts directly with poly(ADP-ribose) via a solvent-accessible and positively charged patch conserved in the Animalia kingdom but not in Fungi. Thus, ubiquitin activation can anchor to poly(ADP-ribose)-seeded protein assemblies, ensuring the formation of functional ataxia-telangiectasia mutated and RAD3-related-signalling complexes.

Premature activation of the SLX4 complex by Vpr promotes G2/M arrest and escape from innate immune sensing.

The HIV auxiliary protein Vpr potently blocks the cell cycle at the G2/M transition. Here, we show that G2/M arrest results from untimely activation of the structure-specific endonuclease (SSE) regulator SLX4 complex (SLX4com) by Vpr, a process that requires VPRBP-DDB1-CUL4 E3-ligase complex. Direct interaction of Vpr with SLX4 induced the recruitment of VPRBP and kinase-active PLK1, enhancing the cleavage of DNA by SLX4-associated MUS81-EME1 endonucleases. G2/M arrest-deficient Vpr alleles failed to interact with SLX4 or to induce recruitment of MUS81 and PLK1. Furthermore, knockdown of SLX4, MUS81, or EME1 inhibited Vpr-induced G2/M arrest. In addition, we show that the SLX4com is involved in suppressing spontaneous and HIV-1-mediated induction of type 1 interferon and establishment of antiviral responses. Thus, our work not only reveals the identity of the cellular factors required for Vpr-mediated G2/M arrest but also identifies the SLX4com as a regulator of innate immunity.

FANCD2 binds MCM proteins and controls replisome function upon activation of s phase checkpoint signaling.

Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2-MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis.

Multimicroscopic study of curcumin effect on fixed nonmalignant and cancerous mammalian epithelial cells.

The morphology changes, in particular the organization of microtubules in mammalian nonmalignant HMEC 184A1 and cancerous MCF-7 cells during curcumin treatment have been investigated by utilizing multiphoton, fluorescence, and atomic force (AFM) microscopies. Fluorescence microscopy reveals formation of ring-like structures of microtubules circumscribing the nuclear area in HMEC 184A1 cells after treatment, while in MCF-7 cells, no important changes were observed. Topography analyses of fixed HMEC 184A1 and MCF-7 before and after treatment with curcumin were performed using AFM and the effect of the employed cells' fixation method was investigated on MCF-7 cells. Due to its indepth optical sectioning capacity multiphoton microscopy provided valuable complementary information on curcumin's effect on both cells' types. Combining information provided by AFM and optical fluorescence and biphoton microscopes allows us to gain a better understanding of the cells and their curcumin-induced changes, especially for microtubules which are the main target of antitumor chemotherapy treatments. Our multimicroscopic study demonstrates that 6 h incubation with curcumin does not induce significant modifications in the interphase microtubules in the malignant MCF7cell, whereas it has measurable effects on those of the nonmalignant HMEC 184A1 cells, revealing also morphology modifications over the nuclear area of these cells.

Mobilization of plasma cells in healthy individuals treated with granulocyte colony-stimulating factor for haematopoietic stem cell collection.

In mice, the plasma cell (PC) niche in the bone marrow is close to the haematopoietic stem cell (HSC) niche. We investigated whether PCs can be mobilized into the peripheral blood (PB) in healthy donors receiving granulocyte colony-stimulating factor (G-CSF) for the induction of HSC mobilization into the PB. G-CSF increased the count of circulating PCs 6-fold, that of circulating B lymphocytes 4-fold and that of circulating HSCs 44-fold. Mobilized circulating PCs comprised CD138(-) (62·2%) and CD138(+) (37·8%) PCs, the latter being more mature based on increased CD27, CD38 and cytoplasmic immunoglobulin expression. Mobilized PCs had a phenotype close to that of steady-state PB PCs or in vitro generated PCs, but they expressed L-selectin only weakly. Finally, a median value of 0·4 × 10(6) /kg donor PCs - one-thirtieth of the overall PC count in a healthy adult - was grafted into patients, which could contribute to immune memory recovery.

Increased plasma-immune cytokines throughout the high-dose melphalan-induced lymphodepletion in patients with multiple myeloma: a window for adoptive immunotherapy.

High-dose melphalan (HDM) followed by autologous stem cell transplantation (ASCT) is a standard treatment for patients with multiple myeloma. However, lymphocyte reconstitution is impaired after HDM. Recent work has suggested that the lymphopenia period occurring after various immunosuppressive or chemotherapy treatments may provide an interesting opportunity for adoptive antitumor immunotherapy. The objective of this study was to determine an immunotherapy window after HDM and ASCT, evaluating T cell lymphopenia, and measuring circulating immune cytokine concentrations in patients with multiple myeloma. The counts of T cell subpopulations reached a nadir at day 8 post-ASCT (day 10 post-HDM) and recovered by day 30. IL-6, IL-7, and IL-15 plasma levels increased on a median day 8 post-ASCT, respectively, 35-fold, 8-fold, and 10-fold compared with pre-HDM levels (p < or = 0.05). The increases in IL-7 and IL-15 levels were inversely correlated to the absolute lymphocyte count, unlike monocyte or myeloid counts. Furthermore, we have shown that CD3 T cells present in the ASC graft are activated, die rapidly when they are cultured without cytokine in vitro, and that addition of IL-7 or IL-15 could induce their survival and proliferation. In conclusion, the early lymphodepletion period, occurring 4-11 d post-HDM and ASCT, is associated with an increase of circulating immune cytokines and could be an optimal window to enhance the survival and proliferation of polyclonal T cells present in the ASC autograft and also of specific antimyeloma T cells previously expanded in vitro.

An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization.

Human plasma cells (PCs) and their precursors play an essential role in humoral immune response but are rare and difficult to harvest. We report the generation of human syndecan-1(+) and immunoglobulin secreting PCs starting from memory B cells in a 3-step and 10-day (D) culture, including a 6-fold cell amplification. We report the detailed phenotypic and Affymetrix gene expression profiles of these in vitro PCs as well as of intermediate cells (activated B cells and plasmablasts) compared with memory B cells and bone marrow PCs, which is accessible through an open web ATLAS (http://amazonia.transcriptome.eu/). We show this B cell-to-PC differentiation to involve IRF4 and AICDA expressions in D4 activated B cells, decrease of PAX5 and BCL6 expressions, and increase in PRDM1 and XBP1 expressions in D7 plasmablasts and D10 PCs. It involves down-regulation of genes controlled by Pax5 and induction of genes controlled by Blimp-1 and XBP1 (unfold protein response). The detailed phenotype of D10 PCs resembles that of peripheral blood PCs detected after immunization of healthy donors. This in vitro model will facilitate further studies in PC biology. It will likewise be helpful to study PC dyscrasias, including multiple myeloma.

Heparanase influences expression and shedding of syndecan-1, and its expression by the bone marrow environment is a bad prognostic factor in multiple myeloma.

The heparan sulfate (HS) proteoglycan, syndecan-1, plays a major role in multiple myeloma (MM) by concentrating heparin-binding growth factors on the surface of MM cells (MMCs). Using Affymetrix microarrays and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we show that the gene encoding heparanase (HPSE), an enzyme that cleaves HS chains, is expressed by 11 of 19 myeloma cell lines (HMCLs). In HSPE(pos) HMCLs, syndecan-1 gene expression and production of soluble syndecan-1, unlike expression of membrane syndecan-1, were significantly increased. Knockdown of HPSE by siRNA resulted in a decrease of syndecan-1 gene expression and soluble syndecan-1 production without affecting membrane syndecan-1 expression. Thus, HPSE influences expression and shedding of syndecan-1. Contrary to HMCLs, HPSE is expressed in only 4 of 39 primary MMC samples, whereas it is expressed in 36 of 39 bone marrow (BM) microenvironment samples. In the latter, HPSE is expressed at a median level in polymorphonuclear cells and T cells; it is highly expressed in monocytes and osteoclasts. Affymetrix data were validated at the protein level, both on HMCLs and patient samples. We report for the first time that a gene's expression mainly in the BM environment (ie, HSPE) is associated with a shorter event-free survival of patients with newly diagnosed myeloma treated with high-dose chemotherapy and stem cell transplantation. Our study suggests that clinical inhibitors of HPSE could be beneficial for patients with MM.