Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies.

Primary tumor

10.1038/cdd.2015.165

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Citations (49)

Morphological Assessment of Apoptosis in Human Lung Cells

Humana Press eBooks (2003)

Lori D. Dwyer‐Nield David Dinsdale John Cohen Margaret Squier Alvin M. Malkinson

AbstractApoptosis, a unique mode of cell death that occurs physiologically as part of a "program" to eliminate unwanted cells, was first described in 1972 (1) and is now one of the most active areas of biologic research. This process occurs during development, as a defense mechanism when cells are damaged by disease or toxins, and as a major constituent of normal homeostatic maintenance. Many biochemical assays have been developed to detect and quanitify apoptosis, but altered nuclear morphology is the most reliable feature. KeywordsAcridine OrangePropylene OxideSodium CacodylateFume HoodNuclear MorphologyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Homeostasis

10.1385/1-59259-323-2:283

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Citations (3)

Protein Domains, Catalytic Activity, and Subcellular Distribution of Neuropathy Target Esterase in Mammalian Cells

Journal of Biological Chemistry (2003)

Yong Li David Dinsdale Paul Glynn

Neuropathy target esterase (NTE), the human homologue of a protein required for brain development in Drosophila, has a predicted amino-terminal transmembrane helix (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. Here we describe the expression, in COS cells, of green fluorescent protein-tagged constructs of NTE and mutant proteins lacking the TM or the R- or C-domains. Esterase assays and Western blots of particulate and soluble fractions indicated that neither the TM nor R-domain is essential for NTE catalytic activity but that this activity requires membrane association to which the TM, R-, and C-domains all contribute. Experiments involving proteinase treatment revealed that most of the NTE molecule is exposed on the cytoplasmic face of membranes. In cells expressing a moderate level of NTE and all cells expressing DeltaC-NTE, fluorescence was distributed in an endoplasmic reticulum (ER)-like pattern. Cells expressing high levels of NTE showed aberrant distribution of ER marker proteins and accumulation of NTE on the cytoplasmic surface of ER-derived tubuloreticular aggregates. Deformation of the ER was also seen in cells expressing DeltaR-NTE or enzymatically inactive S966A-NTE but not DeltaTM-NTE. The data suggest that NTE is anchored in the ER via its TM, that its R- and C-domains also interact with the cytoplasmic face of the ER, and that overexpression of NTE causes ER aggregation via intermolecular association of its C-domains.

Esterase

10.1074/jbc.m210743200

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Citations (92)

Selective injury to rat liver Kupffer cells caused by beryllium phosphate: an explanation of reticuloendothelial blockade.

PubMed (1981)

David Dinsdale David N. Skilleter A. A. Seawright

The i.v. administration of suspensions of beryllium phosphate (5-50 mumol/kg) to rats resulted in the vacuolation of hepatic Kupffer cells within 3 h. After 6 h necrotic Kupffer cells were common throughout the sinusoids of the liver but no changes were detected in the hepatic parenchymal cells during this period. A significant reduction in the numbers of intrasinusoidal cells was observed 14 h after treatment but this population had reverted to normal within 24 h. The administration of colloidal carbon to treated animals at this time did, however, demonstrate a reduction in the complement of functional endocytic cells. These results demonstrate a selective destruction of endocytic cells in the liver by this particulate toxin and the limited response by the organ to this injury. These observations are the most probable explanation for the reticuloendothelial blockade known to be caused in vivo by beryllium phosphate.

Mononuclear phagocyte system

Kupffer cell

Parenchyma

Liver cytology

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Citations (6)

TAp63 and ΔNp63 in Cancer and Epidermal Development

Cell Cycle (2007)

Eleonora Candi David Dinsdale Allesandro Rufini Paolo Salomoni Richard A. Knight

The epidermis is a multilayered stratified epithelium, continuously regenerated by differentiating keratinocytes, that requires the transcription factor p63 for its development and maintenance. The TP63 gene encodes two major protein isoforms, TAp63 and DeltaNp63, which have both transactivating and transcriptional repressing activities and regulate a wide range of target genes. TAp63 shows clear pro-apoptotic activity, mediated both by death receptors (CD95, TNF, TRAIL) and mitochondrial (bax, puma) pathways. Conversely, DeltaNp63 protects from apoptosis by directly competing for TAp63 target promoters or sequestering it, forming inactive tetramers. Accordingly, p63 is expressed in epithelial tumors, contributing to both tumorigenesis and chemoresistance. However, the predominant physiological role of p63 is in epithelial development, as demonstrated by the lack of epidermis and other epithelia in p63-deficient mice. The specific role of TAp63 and isoforms in epithelial development remains mostly unclear. Nevertheless, recent work utilizing in vivo genetic complementation of TAp63 and/or DeltaNp63 into a p63 null background has shed new light into the specific functions of the two isoforms and allowed the in vivo validation of several p63 transcriptional targets, originally identified by microarray analysis in in vitro systems. However, despite concerted efforts to address the role of p63 isoforms, several questions remain unanswered. The main aim of this review is to critically discuss the data available in the literature and thoroughly analyze the models proposed.

Epidermis (zoology)

10.4161/cc.6.3.3797

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Citations (183)

An interleukin‐1β‐converting enzyme‐like protease is a common mediator of apoptosis in thymocytes

FEBS Letters (1995)

Howard O. Fearnhead David Dinsdale Gerald M. Cohen

Apoptosis was induced in thymocytes using diverse stimuli in order to identify events within a common apoptotic pathway. Benzyloxycarbonyl‐valinyl‐alaninyl‐aspartyl fluoromethyl ketone (Z‐VAD.FMK), an interleukin‐1β‐converting enzyme (ICE)‐like protease inhibitor, inhibited apoptosis assessed by flow cytometry, proteolysis of poly (ADP)‐ribose polymerase (PARP), an early biochemical marker of apoptosis, and cleavage of DNA to both large kilobase pair fragments.(30–50 and 200–300 kbp) and to nucleosomal fragments. Z‐VAD.FMK also blocked all the classical ultrastructural features of apoptosis including chromatin condensation to one pole of the nucleus, nucleolar disintegration and cytoplasmic vacuolation. These results suggest the involvement of an ICE‐like protease as a common mediator of apoptosis in thymocytes.

Proteolysis

10.1016/0014-5793(95)01228-7

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Citations (102)