Tumor suppressor role of phospholipase Cε in Ras-triggered cancers

Phospholipase Cε (PLCε) has been characterized as a direct effector of Ras in vitro and in cellular systems; however, the role of PLCε in tumorigenesis and its link to Ras in this context remain unclear. To assess the role of PLCε in Ras-driven cancers, we generated two new mouse strains: one carrying a targeted deletion of Plce (Plce^−/−) and the other carrying mutant alleles of Plce unable to bind to Ras (Plce^RAm/RAm). The Plce^−/− and, to a lesser degree, Plce^RAm/RAm transgenic mice exhibited increased susceptibility to tumor formation in the two-stage skin carcinogenesis protocol, revealing a tumor suppressor function for this PLC. This result also suggests that in this context Ras binding in part regulates functions of PLCε. Although significant differences were not seen in the LSL-Kras^G12D nonsmall cell lung carcinoma model, down-regulation of PLCε was found in animal tumors and in cellular systems following expression of the oncogenic Ras. An inhibitory impact of PLCε on cell growth requires intact lipase activity and is likely mediated by protein kinase C enzymes. Further cellular studies suggest involvement of histone deacetylase in the mechanism of PLCε down-regulation. Taken together, our results show a previously unidentified tumor suppressor role for this PLC in animal models and, together with observations of marked down-regulation in colorectal, lung, and skin tumors, suggest its use as a biological marker in cancer.Activating mutations in the Ras subfamily of small GTPases contribute to the formation of a large proportion of human tumors (1). However, selective treatment for Ras oncogenes is not yet available in the clinic and components that could mediate generation and progression of tumors by Ras are being extensively assessed as alternative targets. These components include direct signaling effectors (such as Raf-kinase, PI3-K, and RalGDS) (2–4) and recently identified Ras-binding protein (PDEδ) (5), as well as components less directly linked to Ras or necessary for Ras oncogene-dependent cancer cell survival (for example, Ral, Cdk4, GATA, TBK1) (6–9). Approaches for evaluating their role in vivo are based on mouse models for Ras-triggered cancers. In addition to a two-stage chemical carcinogenesis model where a carcinogen [DMBA, 7.12-dimethylbenz(a)anthracene] generates activating mutations in Hras, other models, including lung and pancreatic cancer, have been developed based on inducible expression of the Kras oncogene (10, 11). By applying these strategies and models to strains with ablated expression of a specific component or using specific inhibitors, it was possible to assess the requirement or contribution of each component in Ras-triggered malignancies. Some of the well-known direct effectors of Ras have been found necessary for tumor formation; in their absence, tumor generation and progression is attenuated or absent. Examples include the requirement for c-Raf in Kras oncogene-driven nonsmall cell lung carcinoma (NSCLC) (4), PI3K p110α in NSCLC, and in two-stage skin cancerogenesis (3) and RalGDS in the same Hras oncogene-driven skin cancer model (2). In some instances, for example for PI3K, it was also possible to assess the requirement for a Ras-binding domain (RA) by generating alleles encoding p110α variants deficient in Ras-binding (3). However, not all proteins implicated in direct binding to Ras appear to have a positive role in Ras oncogene-driven tumors or tumors caused by other factors. For example, depletion of some members of the Ras Association Doman Family (rassf), such as Rassf1A, enhanced tumor formation in mice (12).Phospholipase Cε (PLCε) is a multifunctional signaling protein, incorporating both PLC and guanine nucleotide exchange factor activities (13, 14). Importantly, and in common to several direct effectors of Ras, PLCε has an RA domain that binds several Ras GTPases, including oncogenic Kras and Hras (15). The main insights into in vivo functions of PLCε have been obtained using two different transgenic mouse strains, one with an in-frame deletion within the PLC catalytic domain (Plce1^ΔX/ΔX) and the other lacking PLCε expression (Plce1^−/−) (16). Although in both cases mice were viable, some notable differences in their phenotype, in particular related to heart morphology, have been observed. In assessing functions related to cancer, however, only the Plce1^ΔX/ΔX transgene—where the link between PLCε and a phenotype could be more difficult to interpret—has been used so far (16, 17). Some of these studies have suggested that the function of PLCε could be required for immune responses associated with the skin chemical cancerogenesis and colorectal models (18, 19). However, in the case of another skin cancer, caused by UVB light—which also triggers inflammation (20)—such an overall positive role of PLCε was not seen. Furthermore, several studies based on analysis of human colorectal tumors (21–23) show markedly reduced levels of PLCε and indicate cancer-suppressing functions of PLCε rather than a positive role in generation and progression of tumors. Because of these apparent discrepancies, it is necessary to further scrutinize the role of PLCε in a more comprehensive study, including use of different Plce1 transgenes.We describe herein generation of two unique Plce1 transgenic strains and their analysis in Ras-triggered cancers that, together with cellular studies and information for expression in human tumors, provide evidence for a tumor suppressor role of PLCε.     

Epilepsy surgery for people with a low IQ

Epilepsy is more prevalent in people with a low IQ than the general population. Up to 1 in 4 people with epilepsy may have a learning disability, yet this group are often under-represented in specialist neurology services. This study examined access and outcomes in epilepsy surgery for people with a low IQ in an audit of 953 patients who have undergone pre-surgical evaluation in our service from 1988 to 2007. We noted a significant increase in the proportion of people with an IQ of 70 or less who have been evaluated for surgery since the routine introduction of high resolution MRI. However candidates with an IQ of 70 or less remain significantly less likely to proceed to surgery; with only 1 in 4 offered surgery compared to 1 in 2 of the candidates with an IQ greater than 70.People with a low IQ who proceeded to surgery achieved comparable postoperative seizure control to their counterparts with an IQ greater than 70. Our findings suggest that special consideration should be given to candidates who are excluded from surgery primarily on the basis of neuropsychological data, since the necessary specialist protocols and appropriate neuropsychological test norms are not widely available for this population.     

Epilepsy & IQ: The clinical utility of the Wechsler Adult Intelligence Scale–Fourth Edition (WAIS–IV) indices in the neuropsychological assessment of people with epilepsy

We examined Wechsler Adult Intelligence Scale–Fourth Edition (WAIS–IV) General Ability Index (GAI) and Full Scale Intelligence Quotient (FSIQ) discrepancies in 100 epilepsy patients; 44% had a significant GAI > FSIQ discrepancy. GAI–FSIQ discrepancies were correlated with the number of antiepileptic drugs taken and duration of epilepsy. Individual antiepileptic drugs differentially interfere with the expression of underlying intellectual ability in this group. FSIQ may significantly underestimate levels of general intellectual ability in people with epilepsy. Inaccurate representations of FSIQ due to selective impairments in working memory and reduced processing speed obscure the contextual interpretation of performance on other neuropsychological tests, and subtle localizing and lateralizing signs may be missed as a result.     

Pertussis immunization in paediatric healthcare workers: Knowledge,attitudes, beliefs,and behaviour

Healthcare workers’ (HCWs) knowledge, attitudes, and beliefs regarding pertussis immunization were assessed and compared to the rate of vaccine uptake. A questionnaire was distributed to employees at a paediatric and maternity tertiary care centre. Respondents were then offered a dose of the tetanus, diphtheria, and acellular pertussis vaccine (Tdap) at a free vaccine clinic. In total, 529 out of 3051 (17%) employees completed the survey and 61 received the Tdap vaccine. Although 76% of participants were willing to be immunized, only 15% presented to the clinic. There is a widespread acceptance of pertussis immunization among paediatric HCWs. Stated intentions may be poorly predictive of behaviour. Education and institutional or public funding may improve vaccine uptake.     

Twitch interpolation in the assessment of the maximum force-generating capacity of the jaw-closing muscles in man

The method of twitch interpolation was employed to study the maximum potential bite forces of humans. Transcutaneous electrical stimuli were applied to parts of one or both masseter muscles in eight volunteers while they bit with a variable but controlled isometric force on a uni-directional force transducer held between the anterior teeth. In all participants the twitch force produced by a single 1-ms pulse, of 25–50 mA intensity, was inversely and linearly related to the voluntary bite force. For each participant the slope of the regression between twitch force and bite force depended on the stimulus intensity and not on whether the stimulus was applied to one or both masseters. Extrapolation of the regression lines to zero twitch force showed that they converged towards a bite-force value that, for any given participant varied only a small amount between different stimulus intensities. For most participants this bite force lay above the maximum that they produced voluntarily; voluntary maximum bites ranged from 153 to 593 N, while the extrapolations predicted a narrower and higher potential range of 282–629 N. It was concluded that, for the masseters at least, there is often spare force-generating capacity which individuals are either unable or not prepared to utilize. This method is non-invasive and may help to define better the maximum bite-force potential of humans.     

Synthesis of a drug-like focused library of trisubstituted pyrrolidines using integrated flow chemistry and batch methods

A combination of flow and batch chemistries has been successfully applied to the assembly of a series of trisubstituted drug-like pyrrolidines. This study demonstrates the efficient preparation of a focused library of these pharmaceutically important structures using microreactor technologies, as well as classical parallel synthesis techniques, and thus exemplifies the impact of integrating innovative enabling tools within the drug discovery process.     

Discovery of 6-substituted indole-3-glyoxylamides as lead antiprion agents with enhanced cell line activity, improved microsomal stability and low toxicity

A series of highly potent indole-3-glyoxylamide based antiprion agents was previously characterized, focusing on optimization of structure-activity relationship (SAR) at positions 1-3 of the indole system. New libraries interrogating the SAR at indole C-4 to C-7 now demonstrate that introducing electron-withdrawing substituents at C-6 may improve biological activity by up to an order of magnitude, and additionally confer higher metabolic stability. For the present screening libraries, both the degree of potency and trends in SAR were consistent across two cell line models of prion disease, and the large majority of compounds showed no evidence of toxic effects in zebrafish. The foregoing observations thus make the indole-3-glyoxylamides an attractive lead series for continuing development as potential therapeutic agents against prion disease.     

Cognitive phenotype of juvenile absence epilepsy: An investigation of patients and unaffected siblings

Objective

The cognitive profile of juvenile absence epilepsy (JAE) remains largely uncharacterized. This study aimed to: (1) elucidate the neuropsychological profile of JAE; (2) identify familial cognitive traits by investigating unaffected JAE siblings; (3) establish the clinical meaningfulness of JAE-associated cognitive traits; (4) determine whether cognitive traits across the idiopathic generalized epilepsy (IGE) spectrum are shared or syndrome-specific, by comparing JAE to juvenile myoclonic epilepsy (JME); and (5) identify relationships between cognitive abilities and clinical characteristics.

Methods

We investigated 123 participants—23 patients with JAE, 16 unaffected siblings of JAE patients, 45 healthy controls, and 39 patients with JME—who underwent a comprehensive neuropsychological test battery including measures within four cognitive domains: attention/psychomotor speed, language, memory, and executive function. We correlated clinical measures with cognitive performance data to decode effects of age at onset and duration of epilepsy.

Results

Cognitive performance in individuals with JAE was reduced compared to controls across attention/psychomotor speed, language, and executive function domains; those with ongoing seizures additionally showed lower memory scores. Patients with JAE and their unaffected siblings had similar language impairment compared to controls. Individuals with JME had worse response inhibition than those with JAE. Across all patients, those with older age at onset had better attention/psychomotor speed performance.

Significance

JAE is associated with wide-ranging cognitive difficulties that encompass domains reliant on frontal lobe processing, including language, attention, and executive function. JAE siblings share impairment with patients on linguistic measures, indicative of a familial trait. Executive function subdomains may be differentially affected across the IGE spectrum. Cognitive abilities are detrimentally modulated by an early age at seizure onset.