Functional consequences of sequence alterations in the ATM gene

The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is a high molecular weight, protein (similar to350 kDa) containing a C-terminal protein kinase domain and a number of other putative domains not yet functionally defined. The majority of ATM gene mutations in A-T patients are truncating, resulting in prematurely terminated products that are highly unstable. Missense mutations within the kinase domain and elsewhere in the molecule alter the stability of the protein and lead to loss of protein kinase activity. Only rarely are patients observed with two missense mutations and this gives rise to a milder disease phenotype. Evidence for a dominant interfering effect on normal ATM kinase activity has been reported in cell lines transfected with missense mutant ATM and in cell lines from some A-T heterozygotes. The dominant negative effect of mutant ATM is manifested by an enhancement of cellular radiosensitivity and may be responsible for the cancer predisposition observed in carriers of ATM missense mutations. In this review, we explore the domain structure of the ATM molecule, sites of interaction with other proteins and the consequences of specific amino acid changes on function. (C) 2003 Elsevier B.V. All rights reserved.

Defining the chemotherapeutic targets of Histone Deacetylase inhibitors

The use of many conventional chemotherapeutic drugs is often severely restricted due to dose-limiting toxicities, as these drugs target the destruction of the proliferating fraction of cells, often with little specificity for tumor cells over proliferating normal body tissue. Many newer drugs attempt to overcome this shortcoming by targeting defective gene products or cellular mechanisms that are specific to the tumor, thereby minimizing the toxicity to normal tissue. Histone deacetylase inhibitors are an example of this type of tumor-directed drug, having significant toxicity for tumors but minimal effects on normal tissue. These drugs can affect the transcriptional program by modifying chromatin structure, but it is not yet clear whether specific transcriptional changes are directly responsible for their tumor-selective toxicity. Recent evidence suggests that transcriptional changes underlie their cytostatic activity, although this is not tumor-selective and affects all proliferating cells. Here we present evidence that supports an alternative mechanism for the tumor-selective cytotoxicity of histone deacetylase inhibitors. The target is still likely to be the chromatin histones, but rather than transcriptional changes due to modification of the transcriptionally active euchromatin, we propose that hyperacetylation and disruption of the transcriptionally inactive heterochromatin, particularly the centromeric heterochromatin, and the inability of tumor cells to cell cycle arrest in response to a specific checkpoint, underlies the tumor-selective cytotoxicity of these drugs.

Chromosomal genotoxicity of nitrobenzene and benzonitrile

In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 muM was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 muM, and no-effect-concentrations were between 0.001 and 0.005 muM. Clearly enhanced MN rates were found at 0.1 muM and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37degreesC was seen above the no-effect-concentration of 2 mM, with an IC50 of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 muM and reaching complete inhibition of motility at 30 muM, whereas benzonitrile up to 200 muM did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 muM. This points to the relevance of interactions with the cellular spindle apparatus.

Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) supports in vitro osteogenesis

Studies have demonstrated that polymeric biomaterials have the potential to support osteoblast growth and development for bone tissue repair. Poly( beta- hydroxybutyrate- co- beta- hydroxyvalerate) ( PHBV), a bioabsorbable, biocompatible polyhydroxy acid polymer, is an excellent candidate that, as yet, has not been extensively investigated for this purpose. As such, we examined the attachment characteristics, self- renewal capacity, and osteogenic potential of osteoblast- like cells ( MC3T3- E1 S14) when cultured on PHBV films compared with tissue culture polystyrene ( TCP). Cells were assayed over 2 weeks and examined for changes in morphology, attachment, number and proliferation status, alkaline phosphatase ( ALP) activity, calcium accumulation, nodule formation, and the expression of osteogenic genes. We found that these spindle- shaped MC3T3- E1 S14 cells made cell - cell and cell - substrate contact. Time- dependent cell attachment was shown to be accelerated on PHBV compared with collagen and laminin, but delayed compared with TCP and fibronectin. Cell number and the expression of ALP, osteopontin, and pro- collagen alpha 1( I) mRNA were comparable for cells grown on PHBV and TCP, with all these markers increasing over time. This demonstrates the ability of PHBV to support osteoblast cell function. However, a lag was observed for cells on PHBV in comparison with those on TCP for proliferation, ALP activity, and cbfa- 1 mRNA expression. In addition, we observed a reduction in total calcium accumulation, nodule formation, and osteocalcin mRNA expression. It is possible that this cellular response is a consequence of the contrasting surface properties of PHBV and TCP. The PHBV substrate used was rougher and more hydrophobic than TCP. Although further substrate analysis is required, we conclude that this polymer is a suitable candidate for the continued development as a biomaterial for bone tissue engineering.

Exploiting novel cell cycle targets in the development of anticancer agents

In this review we provide a brief background on the cell cycle and then focus on two novel and emerging areas of cell cycle research that may prove to have significant relevance to the development of novel anticancer agents. In particular, we review the emerging evidence to suggest that histone deacetylase inhibitors may possess cancer cell-specific cytotoxicity due to their ability to target a novel G2/M checkpoint. We also review the recent literature supporting the proposition that inhibition of E2F activity in epithelial cancer cells may prove to be a useful differentiation therapy that operates via cell cycle-dependent and cell cycle-independent mechanisms.

ATM signaling and genornic stability in response to DNA damage

DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangietasia (A-T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed. (C) 2004 Published by Elsevier B.V.

Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells

Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.

Specific morphological features predictive for the basal phenotype in grade 3 invasive ductal carcinoma of breast

Aims: Cytokeratin (CK) 14, a myoepithelial marker, is also expressed in a proportion of breast carcinomas. There is evidence that these tumours show a differing metastatic pattern and clinical outcome from other invasive ductal carcinomas (IDCs) and may need different management. Currently, they are not identified in routine practice and no morphological guidelines exist to aid their identification. The aim of this study was to analyse the histological features of CK14+ IDC. Methods and results: A detailed histological review of 453 grade 3 IDCs revealed 88 (19.4%) that expressed CK14. Assessment was made independently by two pathologists using a standardized 'tick-box' proforma covering grade, architectural and cytological features. The results were analysed using logistic regression to identify features that predicted for basal phenotype. Concordance between the two pathologists was fair to good for most parameters (kappa 0.4-0.6). On multiple logistic regression, the basal phenotype was highly significantly associated with the presence of a central scar (P = 0.005), tumour necrosis (P < 0.0001), presence of spindle cells (P = 0.006) or squamous metaplasia (P < 0.0001), high total mitotic count (> 40 per 10 high-power field) (P = 0.0002) and high nuclear-cytoplasmic ratio (P = 0.0002). Conclusions: Specific morphological features are strongly associated with basal-like breast carcinoma. These could be used in routine diagnostic practice to identify this important subset of tumours.

Inhibition of S/G(2) phase CDK4 reduces mitotic fidelity

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.

Involvement of novel autophosphorylation sites in ATM activation

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

Deep hole drilling with twist drills: aspects of the CNC process and its real time monitoring and adaptive control

Deep hole drilling is one of the most complicated metal cutting processes and one of the most difficult to perform on CNC machine-tools or machining centres under conditions of limited manpower or unmanned operation. This research work investigates aspects of the deep hole drilling process with small diameter twist drills and presents a prototype system for real time process monitoring and adaptive control; two main research objectives are fulfilled in particular : First objective is the experimental investigation of the mechanics of the deep hole drilling process, using twist drills without internal coolant supply, in the range of diarneters Ø 2.4 to Ø4.5 mm and working length up to 40 diameters. The definition of the problems associated with the low strength of these tools and the study of mechanisms of catastrophic failure which manifest themselves well before and along with the classic mechanism of tool wear. The relationships between drilling thrust and torque with the depth of penetration and the various machining conditions are also investigated and the experimental evidence suggests that the process is inherently unstable at depths beyond a few diameters. Second objective is the design and implementation of a system for intelligent CNC deep hole drilling, the main task of which is to ensure integrity of the process and the safety of the tool and the workpiece. This task is achieved by means of interfacing the CNC system of the machine tool to an external computer which performs the following functions: On-line monitoring of the drilling thrust and torque, adaptive control of feed rate, spindle speed and tool penetration (Z-axis), indirect monitoring of tool wear by pattern recognition of variations of the drilling thrust with cumulative cutting time and drilled depth, operation as a data base for tools and workpieces and finally issuing of alarms and diagnostic messages.

Wear of high aluminium zinc-based alloys in plain bearing applications

Two zinc-based alloys of high aluminium content, Super Cosmal alloy containing 60% Al, 6% Si, 1% Cu, 0.3% Mn and HAZCA alloy containing 60% Al, 8% Si, 2% Cu, 0.06% Mg were produced by sand casting. Foundry characteristics in particular, fluidity, mode of solidification and feeding ability were examined. Metallographic analysis of structures was carried out using optical and scanning electron microscopy and their mechanical properties were determined using standard techniques. Dry wear characteristics were determined using a pin-on-disc test, and boundary-lubricated wear was studied using full bearing tests. Results from casting experiments were evaluated and compared with the behaviour of a standard ZA-27 alloy and those from tribological tests with both ZA-27 alloy and a leaded tin-bronze (SAE660) under the same testing conditions. The presence of silicon was beneficial, reducing the temperature range of solidification, improving feeding efficiency and reducing gravity segregation of phases. Use of chills and melt degassing was found necessary to achieve soundness and enhanced mechanical properties. Dry wear tests were performed against a steel counterface for sliding speeds of 0.25, 0.5, 1.0 and 2 m/s and for a range of loads up to 15 kgf. The high aluminium alloys showed wear rates as low as those of ZA-27 at speeds of 0.25 and 0.5 m/s for the whole range of applied loads. ZA-27 performed better at higher speeds. The build up of a surface film on the wearing surface of the test pins was found to be responsible for the mild type of wear of the zinc based alloys. The constitution of the surface film was determined as a complex mixture of aluminium, zinc and iron oxides and metallic elements derived from both sliding materials. For full bearing tests, bushes were machined from sand cast bars and were tested against a steel shaft in the presence of a light spindle oil as the lubricant. Results showed that all zinc based alloys run-in more rapidly than bronze, and that wear in Super Cosmal and HAZCA alloys after prolonged running were similar to those in ZA-27 bearings and significantly smaller than those of the bronze.

Apoptotic and necrotic effects of hexanedione derivatives on the human neuroblastoma line SK-N-SH

The potential cytotoxicity of two hexanedione food additives (2,3 and 3,4 isomers) was evaluated in comparison with the neurotoxic hexane metabolite 2,5-hexanedione in the human SK-N-SH neuroblastoma line using the MTT assay to indicate mitochondrial dehydrogenase activity and flow cytometry to monitor the cell cycle over 48 h. The IC50s of the 2,3-hexanedione (3.3 ± 0.1 mM) and 3,4-hexanedione (3.5 ± 0.1 mM), indicated that the sensitivity of the cells was approximately seven-fold greater to these toxins compared with the 2,5 derivative (IC50 of 22.4 ± 0.2 mM). Comparison between the respective IC50s of the 2,3-hexanedione and 3,4-hexanedione revealed no difference between the two isomers in terms of their effects on MTT turnover. With flow cytometry analysis, all three hexanediones showed increases in apoptosis within their respective concentration ranges of toxicity shown previously by MTT. In the presence of 2,5-hexanedione, between 8.5 and 17 mM concentrations, there was a significant increase in apoptotic nucleoids which was accompanied by a significant fall in the percentage of nucleoids in the G0/G1 phase (72.4 ± 0.3-45.3 ± 0.6%,), and a rise in the numbers of cells in the G2/M phase. This is likely to indicate growth arrest at cell cycle G2/M checkpoint in response to toxin damage. G2/M accumulation was also shown with 3,4 and 2,3 HD, which was maximal at much lower concentrations (approximately 4 and 3 mM, respectively). Arrest at G1 and G2/M phase is indicative of inhibition of the cell cycle at the stages of DNA replication and chromosome segregation, respectively. It was also apparent that flow cytometry, rather than the MTT assay, did distinguish between the effects of the α-diketones 2,3-hexanedione and 3,4-hexanedione on the cell cycle. At a concentration of 5.8 mM 3,4-hexanedione, the percentage of apoptotic nucleoids was 10.9 ± 0.8% whilst apoptosis induced by 3,4-hexanedione had already reached a maximal level of 60.4 ± 0.5%. In summary, flow cytometry indicated that the 3,4-hexanedione derivative was more toxic than its 2,3 isomer and that both food additives caused interruption in the neuroblastoma cell cycle and further investigation may be required to assess if these α-diketones present in diets pose any possible risks to human health. © 2006 Elsevier Ireland Ltd. All rights reserved.