Anti proliferative activity of ELACYT™ (CP-4055) in combination with cloretazine (VNP40101M), idarubicin, gemcitabine, irinotecan and topotecan in human leukemia and lymphoma cells

This study evaluated combination drug partners for CP-4055, the C18:1Δ9,trans unsaturated fatty acid ester of cytarabine in HL-60 and U937 cells. Growth inhibition was assessed by ATP assay and drug interaction by the combination index and three dimensional methods. Synergy was observed in HL-60 cells for simultaneous combinations of CP-4055 with gemcitabine, irinotecan and topotecan, while combinations with cloretazine (VNP40101M) and idarubicin were additive. In U937 cells, synergy was observed with gemcitabine and additivity for the other drugs. In HL-60, the IC50 concentration of CP-4055 could be reduced 10-fold and that of gemcitabine 3-fold in combination versus the agents alone, an interaction that was independent of drug sequence, ratio and exposure time. In contrast, interactions of CP-4055 with the topoisomerase inhibitors became antagonistic when the drugs were administered 24 h prior to CP-4055 and at certain drug ratios, particularly in U937 cells. In summary, CP-4055 produced additive to synergistic anti proliferative activity when combined simultaneously with drugs from four mechanistic classes in cell culture models of human leukemia and lymphoma. The impact of drug sequence and ratio on the interactions argues for incorporation of these parameters into the design of combination chemotherapy regimens

Cellular Radiosensitivity: How much better do we understand it?

Purpose: Ionizing radiation exposure gives rise to a variety of lesions in DNA that result in genetic instability and potentially tumorigenesis or cell death. Radiation extends its effects on DNA by direct interaction or by radiolysis of H2O that generates free radicals or aqueous electrons capable of interacting with and causing indirect damage to DNA. While the various lesions arising in DNA after radiation exposure can contribute to the mutagenising effects of this agent, the potentially most damaging lesion is the DNA double strand break (DSB) that contributes to genome instability and/or cell death. Thus in many cases failure to recognise and/or repair this lesion determines the radiosensitivity status of the cell. DNA repair mechanisms including homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to protect cells against DNA DSB. Mutations in proteins that constitute these repair pathways are characterised by radiosensitivity and genome instability. Defects in a number of these proteins also give rise to genetic disorders that feature not only genetic instability but also immunodeficiency, cancer predisposition, neurodegeneration and other pathologies. Conclusions: In the past fifty years our understanding of the cellular response to radiation damage has advanced enormously with insight being gained from a wide range of approaches extending from more basic early studies to the sophisticated approaches used today. In this review we discuss our current understanding of the impact of radiation on the cell and the organism gained from the array of past and present studies and attempt to provide an explanation for what it is that determines the response to radiation

Involvement of circulating CEA in liver metastases from colorectal cancers re-examined in a new experimental model

Both experimental and clinical data show evidence of a correlation between elevated blood levels of carcinoembryonic antigen (CEA) and the development of liver metastases from colorectal carcinomas. However, a cause-effect relationship between these two observations has not been demonstrated. For this reason, we developed a new experimental model to evaluate the possible role of circulating CEA in the facilitation of liver metastases. A CEA-negative subclone from the human colon carcinoma cell line CO115 was transfected either with CEA-cDNA truncated at its 3' end by the deletion of 78 base pairs leading to the synthesis of a secreted form of CEA or with a full-length CEA-cDNA leading to the synthesis of the entire CEA molecule linked to the cell surface by a GPI anchor. Transfectants were selected either for their high CEA secretion (clone CO115-2C2 secreting up to 13 microg CEA per 10(6) cells within 72 h) or for their high CEA membrane expression (clone CO115-5F12 expressing up to 1 x 10(6) CEA molecules per cell). When grafted subcutaneously, CO115-2C2 cells gave rise to circulating CEA levels that were directly related to the tumour volume (from 100 to 1000 ng ml(-1) for tumours ranging from 100 to 1000 mm3), whereas no circulating CEA was detectable in CO115 and CO115-5F12 tumour-bearing mice. Three series of nude mice bearing a subcutaneous xenograft from either clone CO115-2C2 or the CO115-5F12 transfectant, or an untransfected CO115 xenograft, were further challenged for induction of experimental liver metastases by intrasplenic injection of three different CEA-expressing human colorectal carcinoma cell lines (LoVo, LS174T or CO112). The number and size of the liver metastases were shown to be independent of the circulating CEA levels induced by the subcutaneous CEA secreting clone (CO115-2C2), but they were directly related to the metastatic properties of the intrasplenically injected tumour cells

A Newly Identified Essential Complex, Dre2-Tah18, Controls Mitochondria Integrity and Cell Death after Oxidative Stress in Yeast

A mutated allele of the essential gene TAH18 was previously identified in our laboratory in a genetic screen for new proteins interacting with the DNA polymerase delta in yeast [1]. The present work shows that Tah18 plays a role in response to oxidative stress. After exposure to lethal doses of H2O2, GFP-Tah18 relocalizes to the mitochondria and controls mitochondria integrity and cell death. Dre2, an essential Fe/S cluster protein and homologue of human anti-apoptotic Ciapin1, was identified as a molecular partner of Tah18 in the absence of stress. Moreover, Ciapin1 is able to replace yeast Dre2 in vivo and physically interacts with Tah18. Our results are in favour of an oxidative stress-induced cell death in yeast that involves mitochondria and is controlled by the newly identified Dre2-Tah18 complex

Standardized and reproducible methodology for the comprehensive and systematic assessment of surgical resection margins during breast-conserving surgery for invasive breast cancer

<p>Abstract</p> <p>Background</p> <p>The primary goal of breast-conserving surgery (BCS) is to completely excise the tumor and achieve "adequate" or "negative" surgical resection margins while maintaining an acceptable level of postoperative cosmetic outcome. Nevertheless, precise determination of the adequacy of BCS has long been debated. In this regard, the aim of the current paper was to describe a standardized and reproducible methodology for comprehensive and systematic assessment of surgical resection margins during BCS.</p> <p>Methods</p> <p>Retrospective analysis of 204 BCS procedures performed for invasive breast cancer from August 2003 to June 2007, in which patients underwent a standard BCS resection and systematic sampling of nine standardized re-resection margins (superior, superior-medial, superior-lateral, medial, lateral, inferior, inferior-medial, inferior-lateral, and deep-posterior). Multiple variables (including patient, tumor, specimen, and follow-up variables) were evaluated.</p> <p>Results</p> <p>6.4% (13/204) of patients had positive BCS specimen margins (defined as tumor at inked edge of BCS specimen) and 4.4% (9/204) of patients had close margins (defined as tumor within 1 mm or less of inked edge but not at inked edge of BCS specimen). 11.8% (24/204) of patients had at least one re-resection margin containing additional disease, independent of the status of the BCS specimen margins. 7.1% (13/182) of patients with negative BCS specimen margins (defined as no tumor cells seen within 1 mm or less of inked edge of BCS specimen) had at least one re-resection margin containing additional disease. Thus, 54.2% (13/24) of patients with additional disease in a re-resection margin would not have been recognized by a standard BCS procedure alone (P < 0.001). The nine standardized resection margins represented only 26.8% of the volume of the BCS specimen and 32.6% of the surface area of the BCS specimen.</p> <p>Conclusion</p> <p>Our methodology accurately assesses the adequacy of surgical resection margins for determination of which individuals may need further resection to the affected breast in order to minimize the potential risk of local recurrence while attempting to limit the volume of additional breast tissue excised, as well as to determine which individuals are not realistically amendable to BCS and instead need a completion mastectomy to successfully remove multifocal disease.</p

Nucleolin Inhibits G4 Oligonucleotide Unwinding by Werner Helicase

The Werner protein (WRNp), a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL), an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA).These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes

8-Oxoguanine rearranges the active site of human topoisomerase I

7,8-Dihydro-8-oxoguanine (8-oxoG) is the most common form of oxidative DNA damage in human cells. Biochemical studies have shown that 8-oxoG decreases the DNA cleavage activity of human topoisomerase I, an enzyme vital to DNA metabolism and stability. We present the 3.1-Å crystal structure of human topoisomerase I in noncovalent complex with a DNA oligonucleotide containing 8-oxoG at the +1 position in the scissile strand. We find that 8-oxoG reorganizes the active site of human topoisomerase I into an inactive conformation relative to the structures of topoisomerase I–DNA complexes elucidated previously. The catalytic Tyr-723–Phe rotates away from the DNA cleavage site and packs into the body of the molecule. A second active-site residue, Arg-590, becomes disordered and is not observed in the structure. The docked, inactive conformation of Tyr-723–Phe is reminiscent of the related tyrosine recombinase family of integrases and recombinases, suggesting a common regulatory mechanism. We propose that human topoisomerase I binds to DNA first in an inactive conformation and then rearranges its active site for catalysis. 8-OxoG appears to impact topoisomerase I by stabilizing the inactive, DNA-bound state

Influence of cyclic bending loading on in vivo skeletal tissue regeneration from periosteal origin

International audienceIntroduction: Periosteum osteogenic and chondrogenic properties stimulate the proliferation then differentiation of mesenchymal precursor cells originating from its deeper layers and from neighboring host tissues. The local mechanical environment plays a role in regulating this differentiation of cells into lineages involved in the skeletal regeneration process. Hypothesis: The aim of this experimental animal study is to explore the influence of cyclic high amplitude bending-loading on skeletal tissue regeneration. The hypothesis is that this mechanical loading modality can orient the skeletogenesis process towards the development of anatomical and histological articular structures. Material and methods: A vascularised periosteal flap was transferred in close proximity to each knee joint line in 17 rabbits. On one side, the tibiofemoral joint space was bridged and loading occurred when the animal bent its knee during spontaneous locomotion. On the other side, the flap was placed 12 mm distal to the joint line producing no loading during bending. Tissue regeneration was chronologically analyzed on histologic samples taken from the 4th day to the 6th month. Results: The structure and mechanical behavior of regenerating tissue evolved over time. As a result of the cyclic bending-loading regimen, cartilage tissue was maintained in specific areas of the regenerating tissue. When loading was discontinued, final osteogenic and fibrogenic differentiation occurred in the neoformed cartilage. Fissures developed in the cartilage aggregates resulting in pseudo-gaps suggesting similar processes to embryonic articular development. Ongoing mesenchymal stem cells stimulation was identified in the host tissues contiguous to the periosteal transfer. Discussion: These results suggest that the pseudarthrosis concept should be reconsidered within the context of motion induced articular histogenesis. (C) 2010 Elsevier Masson SAS. All rights reserved