Gareth J. Griffiths

PKC-delta is an apoptotic lamin kinase.

Protein kinase C-δ is activated during apoptosis, following proteolytic cleavage by caspase 3. Furthermore, overexpression of the catalytic kinase fragment of PKC-δ induces the nuclear phenotype associated with apoptosis, though the molecular basis of this effect has not been determined. In these studies we have examined the role of PKC-δ in the disassembly of the nuclear lamina at apoptosis. The nuclear lamina is disassembled during mitosis and apoptosis and mitotic disassembly involves hyperphosphorylation of lamin proteins by mitotic lamin kinases. During apoptosis, lamin proteins are degraded by caspase 6 and the contribution made by phosphorylation has not been proven. We show here that protein kinase C-δ co-localized with lamin B during apoptosis and activation of PKC-δ by caspase 3 was concomitant with lamin B phosphorylation and proteolysis. Inhibition of PKC-δ delayed lamin proteolysis, even in the presence of active caspase 6, whilst inhibitors of mitotic lamin kinases were without effect. In addition recombinant human PKC-δ was able to phosphorylate lamin B in vitro suggesting that its actions are direct and not via an intermediary kinase. We propose that PKC-δ is an apoptotic lamin kinase and that efficient lamina disassembly at apoptosis requires both lamin hyperphosphorylation and caspase mediated proteolysis.

Damage-induced Bax N-terminal change, translocation to mitochondria and formation of Bax dimers/complexes occur regardless of cell fate.

Sequential steps in the activation of the pro‐apoptotic protein Bax are described for cells with different sensitivity to cytotoxins. SH‐EP1 and SH‐SY5Y human neuroblastoma cells, derived from a single precursor cell line, differed in their sensitivity to taxol but showed the same sensitivity to cisplatin. Both drugs, in both cell lines, induced exposure of a constitutively occluded N‐terminal epitope of Bax. This was reversible and occurred before the translocation of cytosolic Bax to mitochondria. The N‐terminal change in Bax, its subsequent movement to mitochondria and its dimerization/complex formation were insufficient for commitment to death, occurring in the same proportion of cells that either maintained (SH‐SY5Y) or lost (SH‐EP1) clonogenic survival after taxol treatment. Suppression of taxol‐induced apoptosis occurred upstream of cytochrome c release from mitochondria in SH‐SY5Y cells. The data suggest that a further drug damage‐induced event occurs after Bax dimerization/complex formation but prior to cytochrome c release. This event was absent in the taxol‐resistant cells.

Cellular damage signals promote sequential changes at the N-terminus and BH-1 domain of the pro-apoptotic protein Bak.

The pro-apoptotic protein Bak is converted from a latent to an active form by damage-induced signals. This process involves an early exposure of an occluded N-terminal epitope of Bak in intact cells. Here we report a subsequent damage-induced change in Bak, detected using an antibody to the central BH-1 domain. Bak co-immunoprecipitated with Bc1-xL both in undamaged cells and early after damage, when the N-terminal epitope was exposed but the BH-1 epitope remained occluded. A subsequent decrease in binding of Bak to Bc1-xL correlated with exposure of an epitope in the Bak BH-1 domain. Overexpression of Bc1-xL did not affect the kinetics of exposure of the Bak N-terminal epitope but delayed exposure of the BH-1 domain. Cytochrome c release from mitochondria facilitates the activation of apoptotic caspases. The majority of cells with exposed Bak BH-1 domains contained cytosolic cytochrome c. However, a small proportion of cells exhibited exposed Bak BH-1 domains that co-localized with mitochondrial cytochrome c. The data are consistent with a two-step model for the activation of Bak by drug-induced damage signals where dissociation of Bc1-xL from the BH-1 domain of Bak occurs immediately prior to or concomitantly with cytochrome c release.

Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line

Butyrate, a known histone deacetylase inhibitor (HDACi) and product of fibre fermentation, is postulated to mediate the protective effect of dietary fibre against colon cancer. The transcription factor Sp1 is a target of acetylation and is known to be associated with class I HDACs, including HDAC1. Sp1 is a ubiquitous transcription factor and Sp1-regulated genes include those involved in cell cycle regulation, apoptosis and lipogenesis: all major pathways in cancer development. The only known acetylated residue of Sp1 is lysine703 which resides in the DNA binding domain. Here we show that acetylated Sp1 loses p21- and bak-promoter -binding function in vitro. Furthermore treatment with a panel of HDAC inhibitors showed clustering of activities for a subset of inhibitors, causing G2 cell cycle arrest, Sp1 acetylation, p21 and Bak over-expression, all with very similar EC50 concentrations. These HDACi activities were not distributed according to the molecular class of compound. In order to mimic loss of binding, an siRNA strategy was used to reduce Sp1 expression. This resulted in altered expression of multiple elements of the p53/p21 pathway. Taken together our data suggest a mechanistic model for the chemopreventive actions of butyrate in colon epithelial cells, and provide new insight into the differential activities some classes of HDAC inhibitors.

Application of high-content analysis to the study of post-translational modifications of the cytoskeleton.

Cytokeratins 8 and 18 have recently been identified as acetylated. The acetylation of other cytoskeletal proteins has been reported as linked to stabilility. As colorectal cells exist bathed in pharmacologically active levels of the HDACi butyrate, we sought to apply state-of-the-art High Content Analytical approaches to identify the effect of butyrate upon the cytoskeleton of colorectal cells. We observed butyrate caused an increase in acetylation at three distinct residues of cytokeratin 8 (K10, K471, and K482) and that the kinetics of altered acetylation were distinct, implying either separate HDACs, or a heirachy of acetylation. This change in acetylation was associated with a breakdown in the cytokeratin cytoskeleton, implying a functional role for cytokeratin acetylation.

Construction and characterization of multiple human colon cancer cell lines for inducibly regulated gene expression

Validation of targets for cancer drug discovery requires robust experimental models. Systems based on inducible gene expression are well suited to this purpose but are difficult to establish in several epithelial cell types. Using the recently discovered transcriptional transactivator (rtTA2S‐M2), we developed a strategy for fast and efficient generation of Tet On cells. Multiple clones of HCT116, SW480, and HT29 human colon cancer cells for doxycycline‐regulated gene expression were constructed that constitutively express green fluorescent protein (GFP) for selection/maintenance purposes. The cell lines displayed good fold inducibility (49–124× HCT116; 178–621× SW480; 261–787× HT29) and minimal leakiness after transient transfection with a luciferase reporter or with vectors driving inducible expression of red fluorescent protein (dsRed2), constitutively active c‐Src or dominant negative K‐Ras4B. The clones preserved their transformed phenotype as demonstrated by comparing their properties to respective wild type cells, in terms of growth in vitro and in vivo (as tumor xenografts), cell cycle traverse, and sensitivity to drugs used in chemotherapy. These engineered cell lines enabled tightly controlled inducible gene expression both in vitro and in vivo, and proved well suited for construction of double‐stable cell lines inducibly expressing a protein of interest. As such they represent a useful research tool for example, to dissect oncogene function(s) in colon cancer. Supplementary material for this article be found at http://www.mrw.interscience.wiley.com/suppmat/0730‐2312/suppmat/94/suppmat_welman.doc.

A Comparison of High-Content Screening versus Manual Analysis to Assay the Effects of Mesenchymal Stem Cell-Conditioned Medium on Neurite Outgrowth In Vitro

Bone marrow mesenchymal stem cells (MSCs) promote nerve growth and functional recovery in animal models of spinal cord injury (SCI) to varying levels. The authors have tested high-content screening to examine the effects of MSC-conditioned medium (MSC-CM) on neurite outgrowth from the human neuroblastoma cell line SH-SY5Y and from explants of chick dorsal root ganglia (DRG). These analyses were compared to previously published methods that involved hand-tracing individual neurites. Both methods demonstrated that MSC-CM promoted neurite outgrowth. Each showed the proportion of SH-SY5Y cells with neurites increased by ~200% in MSC-CM within 48 h, and the number of neurites/SH-SY5Y cells was significantly increased in MSC-CM compared with control medium. For high-content screening, the analysis was performed within minutes, testing multiple samples of MSC-CM and in each case measuring >15,000 SH-SY5Y cells. In contrast, the manual measurement of neurite outgrowth from >200 SH-SY5Y cells in a single sample of MSC-CM took at least 1 h. High-content analysis provided additional measures of increased neurite branching in MSC-CM compared with control medium. MSC-CM was also found to stimulate neurite outgrowth in DRG explants using either method. The application of the high-content analysis was less well optimized for measuring neurite outgrowth from DRG explants than from SH-SY5Y cells.

High content and high throughput screening to assess the angiogenic and neurogenic actions of mesenchymal stem cells in vitro

Mesenchymal stem cells are universally regarded across many fields of medicine, as one of the most promising cell types for use in cell-based therapies. Although not yet fully understood, the therapeutic effects of these cells are largely attributed to the trophic actions of growth factors and cytokines present in the cell secretome. Specifically, the angiogenic and neurogenic properties of these cells make them attractive for the repair of vascularised and innervated tissues. In this study, we investigate the effect of mesenchymal stem cell conditioned media on in vitro assays of angiogenesis and nerve growth. We describe the use of two state of the art high content and high throughput cell analysis systems and compare them against manual analysis techniques. Mesenchymal stem cell secretomes stimulated angiogenesis and nerve growth in vitro in a donor dependant manner. Levels of neuroregulin, platelet-derived growth factor-AA and glial-derived neurotrophic factor, positively correlated with the observed angiogenic effects of these cells. High content and high throughput cell analysis systems such as the ones used in this study, may provide rapid screening tools to assist not only with patient selection but the identification of predictive therapeutic markers to support clinical outcome monitoring for patients treated with stem cell therapies.

Application of High Content Biology to Yield Quantitative Spatial Proteomic Information on Protein Acetylations

High content analysis (HCA; also referred to as high content biology) is a quantitative, automated, medium-throughput microscopy approach whereby cell images are segmented into relevant compartments (nuclei, cytoplasm) and the staining in each compartment quantified by computer algorithms. The extraction of quantitative information from the cell image generates a wealth of data which contributes significantly to the acceleration of drug discovery and biological research. Here we have adapted HCA to analyze protein acetylations in the cytoskeleton. This approach yields associative information on the link between acetylation and cytoskeletal organization. The protocol also describes optimization steps for cytoskeletal analysis and its application across different cell types, and HCA platforms. The methods described herein are readily adaptable to non-cytoskeletal acetylations and have been applied to the analysis of transcription factors.

Abstract 1713: Mechanisms of resistance to platinum based drugs uncovered by protection caused by substituted coumarins

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Disruption of p53 can have a profound effect on cellular sensitivity to Platinum-based drugs. Inhibition of NQO1 by dicoumarol and other substituted coumarins can target p53 for degradation. Thus, we tested the ability of NQO1 inhibitors to protect HCT116 cells (containing wild type p53) against the cytotoxic effects of cisplatin. All the NQO1 inhibitors protect cells against the damaging effects of cisplatin (see the [table][1] for representative data for one of the inhibitors, AS3). Protection was also seen in a second cell line with wild type p53 (RT112 bladder carcinoma cells). To confirm p53 dependence, experiments were carried out using cells with mutant (MDA231) or null (HCT116 -/-) p53. Surprisingly, the coumarin-based agents also protected these cells against cisplatin-induced damage. In addition, these observations were not dependent on NQO1, since MDA231 cells, with negligible NQO1, and cells overexpressing NQO1 (MDA321-DTD) were similarly protected. Protection is also observed for oxaliplatin but, interestingly, there is no protection against etoposide toxicity. Cell cycle analysis of HCT116 cells treated with cisplatin show that, 24 hours after treatment, cells are blocked in G2. When cells are given the dicoumarol analogues at the same time as the cisplatin, no cell cycle delay is observed. Further, analysis of apoptosis mediators shows that treatment with AS3 inhibits oxaliplatin induction of Bak and reduces caspase-3 activity. Understanding the mechanism by which treatment with the dicoumarol analogues can cause resistance to cisplatin could reveal new methods for potentially overcoming resistance to platinum-based drugs in the clinic. ![Figure][2] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1713. doi:10.1158/1538-7445.AM2011-1713 [1]: #F1 [2]: pending:yes