Impact of human bladder cancer cell architecture on autologous T-lymphocyte activation

To investigate the influence of tumor cell architecture on T-cell activation, we used an autologous human model based on 2 bladder tumor cell lines as targets for cytotoxic tumor-infiltrating lymphocytes (TILs). These tumor cell lines were grown in vitro as either standard 2-dimensional (2D) monolayers or 3-dimensional (3D) spheroids. T-cell activation was determined by measuring the production of three major cytokines (tumor necrosis factor, granulocyte/macrophage colony-stimulating factor and interferon-gamma), known to be secreted by most activated TILs. Changes in the architecture of target cells from 2D to 3D induced a dramatic decrease in their capacity for stimulating TILs. Interestingly, neither TIL infiltration nor MHC class I, B7.1 costimulatory or lymphocyte function-associated factor-3 adhesion molecule downregulation played a major role in this decrease. These findings demonstrate that tumor architecture has a major impact on T-cell activation and might be implicated in the escape of tumor cells from the immune system

Anti-tumour activity in vitro and in vivo of selective differentiating agents containing hydroxamate

A series of hydroxamates, which are not metalloprotease inhibitors, have been found to be selectively toxic to a range of transformed and human tumour cells without killing normal cells (fibroblasts, melanocytes) at the same concentrations. Within 24 h of treatment, drug action is characterized by morphological reversion of tumour cells to a more normal phenotype (dendritic morphology), and rapid and reversible acetylation of histone H4 in both tumour and normal cells. Two; hydroxamates inhibited growth of xenografts of human melanoma cells in nude mice; resistance did not develop in vivo or in vitro. A third hydroxamate, trichostatin A, was active in vitro but became inactivated and had no anti-tumour activity in vivo. Development of dendritic morphology was found to be dependent upon phosphatase activity, RNA and protein synthesis. Proliferating hybrid clones of sensitive and resistant cells remained sensitive to ABHA, indicating a dominant-negative mechanism of sensitivity. Histone H4 hyperacetylation suggests that these agents act at the chromatin level. This work may lead to new drugs that are potent, and selective anti-tumour agents with low toxicity to normal Cells

Histone deacetylase inhibitor induces DNA damage, which normal but not transformed cells can repair

Histone deacetylase inhibitors (HDACi) developed as anti-cancer agents have a high degree of selectivity for killing cancer cells. HDACi induce acetylation of histones and nonhistone proteins, which affect gene expression, cell cycle progression, cell migration, and cell death. The mechanism of the tumor selective action of HDACi is unclear. Here, we show that the HDACi, vorinostat (Suberoylanilide hydroxamic acid, SAHA), induces DNA double-strand breaks (DSBs) in normal (HFS) and cancer (LNCaP, A549) cells. Normal cells in contrast to cancer cells repair the DSBs despite continued culture with vorinostat. In transformed cells, phosphorylated H2AX (γH2AX), a marker of DNA DSBs, levels increased with continued culture with vorinostat, whereas in normal cells, this marker decreased with time. Vorinostat induced the accumulation of acetylated histones within 30 min, which could alter chromatin structure-exposing DNA to damage. After a 24-h culture of cells with vorinostat, and reculture without the HDACi, γH2AX was undetectable by 2 h in normal cells, while persisting in transformed cells for the duration of culture. Further, we found that vorinostat suppressed DNA DSB repair proteins, e.g., RAD50, MRE11, in cancer but not normal cells. Thus, the HDACi, vorinostat, induces DNA damage which normal but not cancer cells can repair. This DNA damage is associated with cancer cell death. These findings can explain, in part, the selectivity of vorinostat in causing cancer cell death at concentrations that cause little or no normal cell death

p53 status correlates with histopathological response in patients with soft tissue sarcomas treated using isolated limb perfusion with TNF-α and melphalan

Background: Recombinant tumor necrosis factor-α (TNF-α) combined to melphalan is clinically administered through isolated limb perfusion (ILP) for regionally advanced soft tissue sarcomas of the limbs. In preclinical studies, wild-type p53 gene is involved in the regulation of cytotoxic action of TNF-α and loss of p53 function contributes to the resistance of tumour cells to TNF-α. The relationship between p53 status and response to TNF-α and melphalan in patients undergoing ILP is unknown. Patients and methods: We studied 110 cases of unresectable limbs sarcomas treated by ILP. Immunohistochemistry was carried out using DO7mAb, which reacts with an antigenic determinant from the N-terminal region of both the wild-type and mutant forms of the p53 protein, and PAb1620mAb, which reacts with the 1620 epitope characteristic of the wild-type native conformation of the p53 protein. The immunohistochemistry data were then correlated with various clinical parameters. Results: P53DO7 was found expressed at high levels in 28 patients, whereas PAb1620 was negative in 20. The tumours with poor histological response to ILP with TNF-α and melphalan showed significantly higher levels of p53-mutated protein. Conclusions: Our results might be a clue to a role of p53 protein status in TNF-α and melphalan response in clinical us

Clinical experience with the novel histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid) in patients with relapsed lymphoma

Preclinical studies indicate that vorinostat (suberoylanilide hydroxamic acid or SAHA) inhibits histone deacetylase (HDAC) activity, increases acetylated histones H2a, H2b, H3, and H4, and thereby induces differentiation and apoptosis in a variety of tumour cell lines, including murine erythroleukaemia, human bladder transitional cell carcinoma, and human breast adenocarcinoma. On the basis of these favourable preclinical findings, vorinostat has been selected as a candidate for clinical development with the potential to treat patients with selected malignances, including Hodgkin's disease and non-Hodgkin's lymphomas. Phase I clinical trials in patients with haematological malignances and solid tumours showed that both intravenous (i.v.) and oral formulations of vorinostat are well tolerated, can inhibit HDAC activity in peripheral blood mononuclear cells and tumour tissue biopsies, and produce objective tumour regression and symptomatic improvement with little clinical toxicity. The dose-limiting toxicities (DLT) of i.v. vorinostat were primarily haematologic and were rapidly reversible within 4–5 days of therapy cessation. In contrast, the DLT for oral vorinostat were primarily non-haematologic (including dehydration, anorexia, diarrhoea, fatigue) and were also rapidly reversible, usually within 3 days. Further research is warranted to optimise the dosing schedule for vorinostat, particularly with respect to dose, timing of administration, and duration of therapy, and to fully delineate the mechanism(s) of antitumour effect of vorinostat in various types of malignances. Several phase II studies are currently ongoing in patients with haematological malignances and solid tumours

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.</p> <p>Methods</p> <p>HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity^®Pathway Analysis.</p> <p>Results</p> <p>Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.</p> <p>Conclusion</p> <p>This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.</p

Aerosols in atmospheric chemistry and biogeochemical cycles of nutrients

Atmospheric aerosols have complex and variable compositions and properties. While scientific interest is centered on the health and climatic effects of atmospheric aerosols, insufficient attention is given to their involvement in multiphase chemistry that alters their contribution as carriers of nutrients in ecosystems. However, there is experimental proof that the nutrient equilibria of both land and marine ecosystems have been disturbed during the Anthropocene period. This review study first summarizes our current understanding of aerosol chemical processing in the atmosphere as relevant to biogeochemical cycles. Then it binds together results of recent modeling studies based on laboratory and field experiments, focusing on the organic and dust components of aerosols that account for multiphase chemistry, aerosol ageing in the atmosphere, nutrient (N, P, Fe) emissions, atmospheric transport, transformation and deposition. The human-driven contribution to atmospheric deposition of these nutrients, derived by global simulations using past and future anthropogenic emissions of pollutants, is put into perspective with regard to potential changes in nutrient limitations and biodiversity. Atmospheric deposition of nutrients has been suggested to result in human-induced ecosystem limitations with regard to specific nutrients. Such modifications favor the development of certain species against others and affect the overall functioning of ecosystems. Organic forms of nutrients are found to contribute to the atmospheric deposition of the nutrients N, P and Fe by 20%–40%, 35%–45% and 7%–18%, respectively. These have the potential to be key components of the biogeochemical cycles since there is initial proof of their bioavailability to ecosystems. Bioaerosols have been found to make a significant contribution to atmospheric sources of N and P, indicating potentially significant interactions between terrestrial and marine ecosystems. These results deserve further experimental and modeling studies to reduce uncertainties and understand the feedbacks induced by atmospheric deposition of nutrients to ecosystems

Gene expression profiles of bladder cancers: evidence for a striking effect of in vitro cell models on gene patterns.

In order to assess the effect of in vitro models on the expression of key genes known to be implicated in the development or progression of cancer, we quantified by real-time quantitative PCR the expression of 28 key genes in three bladder cancer tissue specimens and in their derived cell lines, studied either as one-dimensional single cell suspensions, two-dimensional monolayers or three-dimensional spheroids. Global analysis of gene expression profiles showed that in vitro models had a dramatic impact upon gene expression. Remarkably, quantitative differences in gene expression of 2-63-fold were observed in 24 out of 28 genes among the cell models. In addition, we observed that the in vitro model which most closely mimicked in vivo mRNA phenotype varied with both the gene and the patient. These results provide evidence that mRNA expression databases based on cancer cell lines, which are studied to provide a rationale for selection of therapy on the basis of molecular characteristics of a patient\u27s tumour, must be carefully interpreted

Histone modifications as markers of cancer prognosis: a cellular view

Alterations in modifications of histones have been linked to deregulated expression of many genes with important roles in cancer development and progression. The effects of these alterations have so far been interpreted from a promoter-specific viewpoint, focussing on gene–gene differences in patterns of histone modifications. However, recent findings suggest that cancer tissues also display cell–cell differences in total amount of specific histone modifications. This novel cellular epigenetic heterogeneity is related to clinical outcome of cancer patients and may serve as a valuable marker of prognosis